The present thesis is based on ten peer-reviewed publications that were published between 2013 and 2021 and focus on i) the establishment and further development of analytical methods characterizing physiological features and processes in the intestinal tract and ii) the significance of dietary protein and iii) dietary insoluble fiber on the nutritional physiology of poultry. Chapter I comprises three studies aimed at developing and refining laboratory techniques that will provide better insight into the physiological properties and functions of the intestinal tract. Analytical methods will shed light on the intestinal nutrient transport in chickens, the intestinal mucus layer formation in pigs and the distribution and frequency of intraepithelial immune cells in the gut of chickens. Chapter II includes three publications that examined the effect of qualitative or quantitative differences in dietary protein on the nutritional physiology of chickens. In particular, the impact of feeding differently processed grain legumes on immunological, morphological and functional alterations in the gastrointestinal tract of broilers are investigated. Furthermore, studies regarding protein requirements of male dual purpose chickens were performed investigating the effect of dietary protein reduction on digestive physiology and growth performance. Chapter III is based on a series of four publications evaluating the impact of dietary lignocellulose as an insoluble fiber source in poultry nutrition. Feeding experiments with laying hens and broilers are presented investigating the effect of varying dietary lignocellulose concentrations on chicken’s productive performance, digestive physiology and intestinal microbiota. Finally, a review article summarizes and evaluates studies on the effects of lignocellulose in poultry nutrition and compares the results with those observed in feeding trials using traditional insoluble fiber sources. In the general discussion the most important results from Chapters I-III are highlighted and intensively discussed in comparison with previous literature findings. Furthermore, open research questions are identified and future research perspectives addressed.