With the increasing appearance of highly infectious SARS-CoV-2 variants, mass vaccination becomes a priority for governments around the world. Even if vulnerable populations are vaccinated, antivirals are still required to treat those severely affected by the virus. In the past year, clinical trials have been performed on Camostat mesylate, an inhibitor for the host surface serine protease TMPRSS2, as an antiviral to reduce the severity of SARS-CoV-2 infections. As a general inhibitor for serine proteases, long-term use of Camostat mesylate can lead to the suppression of the patients’ adaptive immune system through off-target inhibition of pro-inflammatory cytokines. In this study, we outline the optimization of expressing and purifying active hTMPRSS2 in E. coli (previously thought to be unfeasible), as well as formulating an in silico and in vitro hybrid HTS pipeline to identify small molecule inhibitors that have higher specificity to the active site of TMPRSS2. After experimenting with different fusion expression constructs and Ni-IMAC purification protocols, the autocatalytic conditions to yield active TMPRSS2 were found to require low salt (150 mM NaCl 50 mM Tris pH 8.0) and given at least 72 hours at 4 oC. Following the successful generation of a quality homology model, successful in silico screening results yielded compounds with a maximum of 15-fold higher binding affinity to the active site of TMPRSS2 compared to Camostat. With an in vitro hit rate of 10% after enriching a commercially available compound library (N = 264, 158 compounds), a patented compound (CIVICYXXDIQUPZ-UHFFFAOYSA-N) was found to inhibit TMPRSS2 to the same degree as recognized antiviral Camostat mesylate with higher binding specificity.