The widespread prevalence of colorectal cancer and its high mortality rate emphasize the urgent need for more effective therapies. When developing new drug products, a key aspect is ensuring that sufficiently high concentrations of the active drug are reached at the site of action. Drug transporters and drug-metabolizing enzymes can significantly influence the absorption and local accumulation of drugs in intestinal tissue. To understand how their presence may affect local drug disposition, the protein abundance of multiple drug transporters and drug-metabolizing enzymes was quantified in paired healthy colonic mucosa and colorectal adenocarcinoma tissue from colorectal cancer patients, utilizing mass spectrometry-based targeted proteomics. Statistically significant changes in protein expression were observed for two transporters (MRP1 and BCRP) and three of the studied enzymes (CES1, CES2 and UGT2B17). MRP1 displayed higher levels in cancerous tissue compared to healthy mucosa samples, while BCRP, CES1, CES2 and UGT2B17 showed the opposite. Other proteins of interest which could be quantified in colonic samples were the drug transporters P-gp, MRP3, MRP4, OATP2B1, MCT1 and enzymes CYP4F2, CYP2J2 and UGT1A1. The insights from this study enhance our understanding of the extent to which drug disposition in tumor tissue of colorectal cancer patients could be impacted by drug transporters and drug-metabolizing enzymes and may facilitate a more accurate prediction of local drug concentrations.