Drug therapy is always at risk of over- or underdosing resulting in inefficient treatment or adverse drug reactions. To find an optimal dose of a drug is therefore crucial. However, determination of the “right dose” is challenging due to several reasons as patient-individual factors impacting the pharmacokinetics or pharmacodynamics of a drug (e.g. obesity) or due to the still present knowledge gaps mainly in special patients (e.g. infants) or special conditions (e.g. hemofiltration). Aggravatingly, the available pharmacokinetic information comprises mainly easily accessible matrices as plasma, which is often not the target site for drugs and may differ significantly with regard of the concentration-time courses of a drug. In addition, small inhomogeneities in the dosage forms change the amount of administered drug which may also influence the therapy outcome. The objective of Project I was to evaluate uniformity of dug content and net mass as well as accuracy of content in contemporaneously compounded hydrocortisone capsules as preparation of a subsequently planned clinical trial. Uniformity and accuracy investigations were performed in adaption of testing methods provided by the European Pharmacopeia using a suitable quantification method developed for this project. The vast majority of capsules showed a good uniformity within the tolerable limits but on the other side, some batches contained capsules with maximum deviations of ±27.6% (net mass) and even ±203% (content) from the respective batch mean which would most probably lead to suboptimal treatment. The accuracy investigation revealed across all capsules and batches a by about 11% decreased dose with similar deviations as found for uniformity of content. In addition, a potential correlation of net mass and drug content was assessed but could be shown in just a minority of batches. The aim of Project II was to investigate potential impact factors of microdialysis sample collection on relative recovery values to inform the study design of a clinical trial investigating antibiotic combinations in obese patients. Several in vitro investigations were performed using a standardised in vitro microdialysis system. Combinations of up to 4 drugs and the order in which drugs were microdialysed did not impact relative recovery, but air in the system led to much lower and highly inconsistent relative recovery values. Hence, air should urgently be avoided to obtain reliable and consistent results from microdialysis sampling. Additionally, time until consistency of obtained relative recovery values after a flow rate change was assessed and resulted in recommendations for equilibration times of at least 10 to 15 min depending on the applied flow rate. Also in Project III, in vitro investigations were performed to evaluate potential impact factors using the in vitro microdialysis system. The investigations showed neither an impact of the pH value in surrounding medium and perfusate, nor of diffusion direction (microdialysis or retrodialysis setting) or vancomycin concentration on resulting relative recovery values. As expected, an impact of flow rate on relative recovery values was observed. The results of the in vitro investigations were successfully implemented in the design of a clinical trial determining the feasibility of vancomycin quantification in infants via subcutaneous microdialysis. Study samples were quantified using a previously in the Department developed LC- MS/MS method. The resulting concentrations showed plausible time courses. In addition, no increased risk of adverse events was observed and therefore feasibility of microdialysis sampling in the infants was proven. Project IV aimed to extent a previously developed HPLC method for the quantification of cefuroxime and linezolid from microdialysate to also plasma samples. The final method was applied for the quantification of both drugs in a clinical trial investigating both drugs as perioperative antibiotic prophylaxis treatment. The plasma concentration-time courses were subsequently compared to previously determined microdialysate and interstitial fluid concentrations showing a very good tissue penetration of cefuroxime and an impaired 1 for linezolid, especially in the synovial fluid of the knee. All here presented projects helped to close knowledge gaps in the treatment of endocrine disorders or bacterial infections. Project I supported the subsequent clinical trial and ultimately the approval of a commercial paediatric dosage form lowering the variability of administered hydrocortisone doses and therefore increasing the therapeutic outcome. The in vitro investigations of Project II and III answer basic research questions in the field of microdialysis and increased the quality of obtained data from the clinical trials by optimising the study designs. Finally, the analysis of clinical trial samples in Project III and IV gave a valuable insight into target site pharmacokinetics.