Human serum albumin (HSA) is a critical transport plasma protein accounting for similar to 60% of the total protein content in blood. Remarkably, this protein is also found to display esterase activity. In this study, we apply theoretical studies to elucidate the origin of the esterase-like activity arising from the Sudlow site I. Using MD and QM/MM calculations, we investigate which active site residues are involved in the reaction, and the precise mechanistic sequence of events. Our results suggest Lys199, His242, and Arg257 help give rise to the esterase activity and that the most catalytically efficient active site configuration requires that both Lys199 and Aspirin are in their neutral forms. The abundance of HSA in the body suggests the protein might be a suitable target for the computational guided design of acetyl based pro-drugs of acidic molecules that often displayed limited oral exposure due to their unmasked ionizable substituent.