A study of proteolysis effects on recombinant protein yield was completed using the insect cell (Sf-9)-baculovirus (AcNPV) expression system. Activities of protease and beta-galactosidase (beta-gal), a marker heterologous protein, were assayed at various multiplicities of infection (MOI = 1, 5, and 20) on a time course postinfection. Also, several protein-substrate gel electrophoresis assays were run using gelatin, beta-gal, and bovine serum albumin (BSA) in the gel matrix, to determine the protein specificity of the proteases. The most abundant protease activity (cysteine), found at 49 kDa, degraded all three substrates, pre-and post-infection. Two other protease activities (40 and 36 kDa) appeared on polyacrylamide gel electrophoresis (PAGE) gels after 72 hpi (hours postinfection). In addition, the culture with the highest MOI had the highest beta-gal activity until 72 hpi, when the activity dramatically decreased coincidentally with a 2.5-fold increase in protease activity. This result and the electrophoresis evidence that the protease is specific to beta-gal, indicate that there is a negative correlation between protease activity and recombinant protein yield, These results guide efforts to control product-degrading proteolysis in insect cell-baculovirus expressions systems by harvest timing and the addition of protease inhibitors.