High-pressure processing (HPP) is a technology of growing interest for food preservation, due to its ability to control the activity of degradative enzymes. The effect of three variables (pressure levels of 100, 250, and 400 MPa; pressure holding times of 0, 5, 15, and 30 min; and pressurization rates of 8 and 14 MPa/s) on the activity of the enzymes acid phosphatase, cathepsins (B and D), lipase, and calpains was studied using sea bass fillets as a case study model. Additionally, the effect of HPP on sarcoplasmic proteins was studied by SDS-PAGE and isoelectric focusing electrophoreses. The increase in pressure level and holding time decreased the protein concentration in sarcoplasmic extracts, and also the activity of calpains. As compared to nontreated samples, acid phosphatase activity was lower at 400 MPa, and for cathepsin D lower activities were observed at 100 and 400 MPa. The increase in pressurization rate increased the activity of cathepsin D, lipase, and calpains, although it was not always significant. In contrast, cathepsin B and lipase activities were less affected by HPP treatments. Electrophoresis separation of sarcoplasmic proteins showed that the intensity of many protein bands changed mainly due to pressure level and holding time. The results of this study suggest that HPP causes lysosomes disruption and also denaturation, aggregation, and fragmentation of sarcoplasmic proteins, and this evidence might be related to the decrease in enzymes activity especially at 400 MPa. In conclusion, HPP can be a potential tool to control the activity of degradative enzymes, which might prevent the softening of sea bass muscle due to autolytic reactions.