The residues involved in substrate interaction of the human apical sodium-dependent bile acid transporter (hASBT, SLC10A2) remain undefined. Biochemical modification of conserved cysteine residues has suggested their direct involvement in hASBT function. In the present study, we developed novel methanethiosulfonyl (MTS)-bile salt derivatives and describe their reactivity toward hASBT and its mutants. Endogenous Cys residues were subjected to Ala/Thr scanning mutagenesis and subsequent exposure to affinity inactivators. We show that C51A/T, C105A/T, C144A, and C255A/T are loss-of-function mutations. Additionally, C74A/T cell surface expression was abolished suggesting a role in protein folding and/or trafficking. C270A remained largely unaffected in the presence of 1.0 mM polar and charged MTS reagents (MTSEA, MTSES, and MTSET) and retained function similar to wt-hASBT control. However, in the presence of synthetic cholyl- and chenodeoxycholyl-MTS analogues, C270A displayed a significant decrease in K(T) and J(max). Our findings demonstrate that Cys270 is a highly accessible extracellular residue susceptible to thiol modification in its native form that remains largely unaffected upon mutation to Ala. Consequently, C270A provides an ideal scaffold for cysteine scanning mutagenesis studies. Furthermore, the substantial decrease in ligand affinity and maximal transport capacity of C270A suggest that C270 may potentially impact, although not critically, a putative substrate binding domain of hASBT. Overall, bile acid-MTS conjugates can serve as novel and powerful tools to probe the role of endogenous as well as engineered Cys residues and, ultimately, aid in defining their role in the bile acid binding region(s) of hASBT.