The levels of drug metabolizing enzymes (DMEs) and transporter proteins in the human intestine are pertinent to determine oral drug bioavailability. Despite the paucity of reports on such measurements, it is well recognised that these values are essential for translating in vitro data on drug metabolism and transport to predict drug disposition in gut wall. In the current study, clinically relevant DMEs (cytochrome P450 (CYP) and uridine 5'- diphospho-glucuronosyltransferase (UGT)) and drug transporters were quantified in total mucosal protein preparations from the human jejunum (n = 4) and ileum (n = 12) using
QconCAT-based targeted proteomics. In contrast to previous reports, UGT2B15 and OATP1A2 were quantifiable in all our samples. Overall, no significant disparities in protein expression were observed between jejunum and ileum. Relative mRNA expression for drug transporters did not correlate with the abundance of their cognate protein except for P-gp and OST-α, highlighting the limitations of RNA as a surrogate for protein expression in dynamic tissues with high turnover. Inter-correlations were found within CYP (2C9–2C19 (p = 0.002, R2 = 0.63), 2C9–2J2 (p = 0.004, R2 = 0.40), 2D6–2J2 (p = 0.002, R2 =0.50)) and UGT (1A1–2B7 (p = 0.02, R2 = 0.87)) family of enzymes. There were also correlations between P-gp and several other proteins (OST-α (p < 0.0001, R2 = 0.77), UGT1A6 (p = 0.009, R2 = 0.38) and CYP3A4 (p = 0.007, R2 = 0.30). Incorporating such correlations into building virtual populations is crucial for obtaining plausible characteristics of simulated individuals.