The COVID-19 pandemic has highlighted the need for new antiviral targets, as many of the currently approved drugs have proven ineffective against mitigating SARS-CoV-2 infections. The host transmembrane serine protease TMPRSS2 is a highly promising antiviral target, as it plays a direct role in priming the spike protein before viral entry occurs. Further, unlike other targets such as ACE2, TMPRSS2 has no known biological role. Here we utilize virtual screening to curate large libraries into a focused collection of potential inhibitors. Optimization of a recombinant expression and purification protocol for the TMPRSS2 peptidase domain facilitates subsequent biochemical screening and characterization of selected compounds from the curated collection in a kinetic assay. In doing so, we demonstrate that serine protease inhibitors camostat, nafamostat, and gabexate inhibit through a covalent mechanism. We further identify new non-covalent compounds as TMPRSS2 protease inhibitors, demonstrating the utility of a combined virtual and experimental screening campaign in rapid drug discovery efforts.