The design of experiments (DoE) is one of the quality-by-design tools valued in analytical method development, not only for cost reduction and time effectiveness, but also for enabling analytical method control and understanding via a systematic workflow, leading to analytical methods with built-in quality. This work aimed at using DoE to enhance method understanding for a developed UHPLC enantioseparation of terbutaline (TER), a model chiral drug, and to define quality assurance parameters associated with using chiral mobile phase additives (CMPA). Within a response surface methodology workflow, the effect of different factors on both chiral resolution and retention was screened and optimized using Plackett-Burman and central composite designs, respectively, followed by multivariate mathematical modeling. This study was able to delimit method robustness and elucidate enantiorecognition mechanisms involved in interactions of TER with the chiral modifiers. Among many CMPAs, successful TER enantioresolution was achieved using hydroxypropyl β-cyclodextrin (HP-β-CD) added to the mobile phase as 5.4 mM HP-β-CD in 52.25 mM ammonium acetate. Yet, limited method robustness was observed upon switching between the different tested CMPA, concluding that quality can only be assured with specific minimal pre-run conditioning time with the CMPA, namely 16-column volume (60 min at 0.1 mL/min). For enantiorecognition understanding, computational molecular modeling revealed hydrogen bonding as the main binding interaction, in addition to dipole-dipole inside the CD cavity for the R enantiomer, while the S enantiomer was less interactive.