In this study, the enzyme papain was treated under different conditions of High Voltage Electric Field (HVEF), where three main factors were varied: frequency (0, 180 and 360 Hz), electric field strength (1, 5, and 9 kV / cm), and processing time (10, 20 and 30 minutes). The objective was to determine the changes in the activity of the enzyme and in its structure. The results mainly showed an effect of reduction on the residual activity (RA) of the enzyme papain. The best effect on the RA (47.9%) was achieved under the most intense treatment conditions of EF (9 kV/cm) and time (30 min). The results in the structure analysis of papain by fluorescence spectroscopy showed that HVEF led to denaturation and possibly aggregation of the enzyme. Therefore, HVEF can be considered as an efficient alternative nonthermal technology that can be used for partial enzyme inactivation.