Human placental alkaline phosphatase has been identified as a hydrolase that is significantly overexpressed on the surface of various solid tumor cells, and is therefore a suitable prodrug design target for non‐invasive cancer imaging and therapy. Structure‐based prediction of enzymatic activities is essential for rational prodrug design. We have been probing the catalytic proficiency – (kcat/KM)/kw– of placental alkaline phosphatase toward several widely diverse substrate structures experimentally and correlating these results to in silico predictions that are based on the free energy estimates obtained from docking of each substrate structure with placental alkaline phosphatase. We have found that electrostatic contribution from the tail group is the most crucial factor to determine the catalytic efficiencies of the substrates. The electrostatic contribution and the total binding energy of the tail group are well correlated with catalytic efficiencies (R2 = 0.79 and 0.89, respectively). However, hydrophobic contribution from the tail group does not correlate with the catalytic efficiencies (negative correlation, R2 = 0.27). This supports the prior hypothesis stating that alkaline phosphatase‐mediated differential hydrolysis of its substrates is attributable to the differential interactions with the tail group, determined by the electrostatic contributions from the non‐bridging oxygen atoms. Calculation of the electrostatic potentials within the active site of human placental alkaline phosphatase also suggests that the local positive electrostatic environment may account for its capability to distinguish various substrates. Our study is likely to have immediate implications in the design of prodrugs against human placental alkaline phosphatase and other esterases overexpressed by human tumor cells.