The human apical bile acid transporter (hASBT) in the distal ileum reabsorbs bile acids and is responsible for transporting approximately 95 % of bile acids back to liver for recycling in the enterohepatic pathway. ASBT activity has been linked to multiple disease states, including Crohn’s disease, hypercholesterolemia, cholestasis, and type 2 diabetes. ASBT activity is regulated at the post-translational level by glycosylation, ubiquitination, and S-acylation, which control its translocation to the cell surface and protein stability. Although biochemical studies have suggested that phosphorylation of serine, threonine, and tyrosine may play a role in the regulation of ASBT function, no study has determined where phosphorylation of ASBT occurs and how its phosphorylation level correlates with its activity. In this study, we developed a workflow using parallel reaction monitoring (PRM) targeted mass spectrometry to determine and quantify the stoichiometry of ASBT phosphorylation in the presence of various kinase inhibitors and activators. Our findings provide the first evidence of ASBT phosphorylation at multiple sites (Thr330, Ser334, and Ser 335), with Ser 335 being the most abundant phosphosite.Futhermore, we demonstrate that the phosphorylation level of Serine 335 coincides with the bile acid uptake activity of ASBT. Finally, we discovered that PKCalpha, but not other PKC isoforms, regulates ASBT phosphorylation at Ser 335. Taken together, our findings establish the molecular basis of phosphorylation-mediated regulation of ASBT which may include novel therapeutic targets for managing ASBT-linked disease.