Enzyme assays that use fluorescently labeled substrates and microplate formats have been incorporated into laboratory protocols to improve sensitivity and reduce the time and labor involved in traditional bench-scale analyses. Microplate protocols vary, and the methods have not been evaluated systematically for comparability and reproducibility. In this study, p-nitrophenol- and 4-methylumbelliferone-based microplate methods for estimating β-glucosidase activity were compared in two soils with different properties. Microplate method reproducibility was evaluated in five replicate soil suspensions, and Michaelis-Menten kinetics for the microplate assays were compared to those of a standard pNP bench-scale assay. The effect of soil sample sonication on reproducibility was determined for the MUF microplate method.  β-glucosidase activity showed significant differences among five replicate soil suspensions for both microplate methods. The Michaelis-Menten kinetic parameter Km was significantly different in the microplate methods compared to the bench method. Enzyme activities measured by the MUF and bench methods were comparable, but the pNP microplate method resulted in more variable measurements, and was less sensitive in the soils studied. Sonication at 15 W mL^-1  prior to suspension of soils resulted in higher (MUF) measurements, but greater variability. The effects of high background absorbance on reproducibility and accuracy do not support the pNP microplate method as a substitute for the standard bench method. A robust comparison study of the MUF microplate method across laboratories is recommended to further validate its use in comparative analyses.