The purpose of the present study was to evaluate the efficacies and mechanisms of the PAB (para-amino benzamidine) affinity column chromatography, virus filtration, pasteurization ($60^{circ}C$ heat treatment for 10 h), and lyophilization steps employed in the manufacture of urokinase from human urine with regard to the removal and/or inactivation of human immuno-deficiency virus (HIV), bovine viral diarrhoea virus (BVDV), bovine herpes virus (BHV), and murine encephalomyocarditis virus (EMCV). Samples from relevant stages of the production process were spiked with each virus and subjected to scale-down processes mimicking the manufacture of urokinase. Samples were collected at each step, immediately titrated using a 50% tissue culture infectious dose $(TCID_{50})$, and the virus reduction factors evaluated. PAB chromatography was found to be an effective step for removing BVDV, BHV, and EMCV with log reduction factors of 2.79,6.50, and 5.96, respectively. HIV, BVDV, BHV, and EMCV were completely removed during the Viresolve NFP filtration step with log reduction factors of ${geq}6.06,;{geq}4.60,;{geq}5.44,;and ;{geq}6.87$, respectively. Pasteurization was also found to be a robust and effective step in inactivating all the viruses tested, since there were no residual viruses detected after the pasteurization process. The log reduction factors achieved by pasteurization were ${geq}5.73$ for HIV, ${geq}3.86$ for BVDV, ${geq}6.75$ for BHV, and ${geq}5.92$ for EMCV. Lyophilization showed significant efficacy for inactivating BVDV, BHV, and EMCV with log reduction factors of 2.69, 1.37, and 4.70, respectively. These results indicate that the production process for urokinase exhibited a sufficient viral reducing capacity to achieve a high margin of virus safety.