The goal of this research was to investigate the effect of different sources and concentrations of trypsin on the viability of bovine sperm as a potential method for removing pathogens similar to the washing methods developed for embryos. Trypsin derived from porcine pancreas (Sigma-Aldrich, St Louis, MO, USA) at 2.5% and 0.25% was compared to the recombinant human sequence (TrypLE Select; Invitrogen, Carlsbad, CA, USA) at 10× and 1× concentrations. Cryopreserved bovine sperm (n = 3 bulls) were thawed and processed using discontinuous (90/45) Percoll density gradient centrifugation; the sperm pellets were then washed (10 min at 300g) in TL-HEPES Solution (Cambrex Corp., East Rutherford, NJ, USA) and then resuspended in 1 mL of the same medium. Aliquots of 200 µL of the washed sperm were then added to 1 mL of each of the 4 trypsin treatments as well as a negative control (without trypsin) and incubated at room temperature. Aliquots (25 µL) of each treatment were examined for progressive motility after 5 min. As a result, the control sperm (no trypsin) increased progressive motility by 6.7% and the 1× TrypLE treatment by 9.3%. However, the 10× TrypLE Select and the 10× and 1× porcine pancreas extracts decreased progressive motility by 8.3, 29.0, and 4.0%, respectively. The objective of the second experiment was to determine if the treatment of bovine semen with trypsin (1× TrypLE Select and 0.25% porcine pancreas extract) before or after cryopreservation would affect sperm quality as compared to cryopreservation without trypsin treatment. Raw semen (n = 6 bulls) was collected, evaluated, cryopreserved, and then thawed using a standard bovine method (Biladyl^®; Minitube, Verona, WI, USA) without further treatment (control) or after treatment with one of two trypsin treatments (density gradient centrifugation with 1× TrypLE Select or 0.25% porcine pancreas extract in the 45% Percoll layer and a soybean trypsin in activator (Sigma) in the 90% layer) either before freezing (Treat–Freeze) or after thawing (Freeze–Treat). The results for the 6 individual bull samples were comparable and are presented as means (± SEM) compared to the cryopreserved control (no trypsin treatment). Using the Mann–Whitney Rank Sum Test, no differences (P > 0.05) were found in any of the parameters comparing the crypreserved controls (no trypsin treatment) and the 4 treatments: Freeze–Treat vs. Treat–Freeze using either the recombinant TrypLE Select (1×) or the porcine pancreas extract (0.25%). These results suggest that trypsin treatment, before or after cryopreservation, can be used safely on bovine sperm without affecting viability in vitro.