Heterotrophic organisms are central to subsurface microbial communities and play an important role in carbon cycling. Most approaches to measuring enzymatic activities rely on the addition of a fluorescently labeled substrate to a sediment incubation. However, quantifying rates of extracellular enzymatic hydrolysis of organic matter is often problematic due to the tendency for a fluorescently labeled organic substrate to sorb to the sediment matrix. This results in lower fluorescence intensities and distorted, inaccurate hydrolysis rate calculations. In this project, a desorption treatment was developed to counteract the adverse effects of sorption on enzymatic activity measurements. Upon subsampling a sediment incubation amended with a fluorescently labeled substrate, the subsample is treated with a concentrated solution of unlabeled substrate, along with 0.2% sodium dodecyl sulfate (SDS), in order to competitively desorb the adsorbed, fluorescent substrate target. This treatment improves measured fluorescence intensities by a median of 62.5%, and is particularly effective at desorbing high molecular weight substrate products, resulting in debiased hydrolysis rates that are 14.75 nM/hr lower on average. Competitive desorption treatment was demonstrated to be effective for multiple substrates and in a broad range of sediments from diverse geological and geochemical contexts. Future applications of this method will result in more quantitative and comparable hydrolysis rates in subsurface sediments, will enable enzymatic activity measurements in problematic sediments that were previously infeasible, and will facilitate physiological characterization of microbial communities and model organisms in order to better understand heterotrophic carbon cycling in the subsurface environment.