Phosphorus cycling in most ecosystems is dependent on plant and microbiologically derived phosphatase enzymes, and available P limits both microbial and plant growth. Measurements of enzymes in soil systems are often time-consuming and labor intensive. In this study, we examined phosphomonoesterase activity in soils using a Zymark XP laboratory robotic system for soil handling, solvent addition and exchange, filtration, incubation, reagent addition, and final sample preparation. Phosphomonoesterase activity was measured using phenyl phosphate as substrate, and samples were analyzed using a 96-well microplate reader. Results indicated that our robotic system was capable of effectively measuring phosphomonoesterase activity in soils differing in physical and chemical characteristics. The results obtained using manual and robotic procedures were comparable in accuracy and precision. The robotic system decreased labor associated with this assay by about a factor of 4.5 relative to the manual system, with considerable savings on reagent costs and labor.