The impact of the intestine in determining the oral bioavailability of drugs has been extensively studied. Its large surface area, metabolic content and positioning at the first site of exposure for orally ingested xenobiotics means its contribution can be significant for certain drugs. However, prediction of the exact metabolic component of the intestine is limited, in part due to limitations in validation of in vitro tools as well as in vitro-in vivo extrapolation scaling factors. Microsomes are a well established in vitro tool for extrapolating hepatic metabolism, however standardised methodologies for preparation in the intestine are limited, in light of complexities in preparation (e.g. presence of multiple non-metabolic cells, proteases and mucus). Therefore, the aims of this study were to establish an optimised method of intestinal microsome preparation via elution in the proximal rat intestine, and to determine microsomal scaling factors by correcting for protein losses during preparation. In addition, to assess species in another preclinical species (dog) and human as well as assessing and regional differences in scaling factors and metabolism. Following optimisation of a reproducible intestinal microsome preparation method in the rat, the importance of heparin in limiting mucosal contamination was established. These microsomes were characterised for total cytochrome P450 (CYP) content, and CYP and uridine 5'-diphosphate glucuronosyltransferase (UGT) activities using maker probes of testosterone and 4-nitrophenol. Loss corrected microsomal scaling factors between two pools of n=9 rats was 9.6±3.5 (recovery 33%). A broad range of compounds (n=25) in terms of metabolic activity and physicochemical properties were screened in rat intestinal microsomes. The prediction accuracy relative to in house generated or literature in vivo estimates of the fraction escaping intestinal metabolism (FG) through in vitro-in vivo extrapolation of observed metabolism and the derived scaling factors and either Caco-2 permeability of physicochemical permeability estimates utilising the Qgut model. In the dog, regional differences in intestinal scaling factors and metabolic activities were explored, as well as relationships between the proximal intestine and liver in matched donors. Positive correlations in both hepatic activity and microsomal scalars were observed. Robust scaling factors were established using the 3 microsomal markers. A total of 24 compounds were screened for hepatic and intestinal metabolism in order to make in vivo estimates of FG, the fraction escaping hepatic metabolism (FH) and oral bioavailability (F). Estimates based on Caco-2 and physicochemical based scaling, as well as utilising a commercial PBPK software platform (ADAM model, Simcyp® v12) were broadly similar with generally reduced prediction accuracy in proximal physicochemical based Qgut scaling, and improved predictions using Caco-2 Qgut or PBPK approaches. Worse predictions were observed for compounds with high protein binding, transporter substrates and/or CYP3A inhibitors. Regional metabolism demonstrated peak metabolism in the proximal intestine, before declining distally. Human intestinal microsomes were prepared for jejunum and ileum tissue. Although samples were limited, regional differences in metabolic activities and scaling factors were also assessed, using correction markers and activity in 23 compounds. In all, 20 compounds overlapped between all three species. Comparison in Fa.FG between rat and human CYP3A substrates showed a modest relationship, however relationships between species and human were generally poor given the observed differing metabolic contributions of testosterone and 4-NP metabolite formation between species limited the observed relationships between species. However, within species, good estimates of oral bioavailability were observed. This is the largest know interspecies comparison of intestinal metabolism and scaling factors with microsomes prepared within the same lab.