Protein hydrolysate is highly regarded as a source of naturally occurring antioxidant peptides. The purpose of this study was to investigate the effect of Ultrasonic (Frequency, 20 KHz; Amplitude, 50%; Time, 30 min) and high-pressure homogenization (Power, 1500 bar; Rated flow, 10 dm/h) pretreatmenton the enzymatic hydrolysis and antioxidant properties of yeast protein hydrolysate obtained from Kluyveromyces marxianus. Trypsin and chymotrypsin were used for protein hydrolysis. Respectively, 73.22%, 23.01% of the total protein was released through sonication and high-pressure homogenization processes. The progress of the enzymatic hydrolysis was evaluated based on the number of free amino groups measured by the O-phetaldialdehyde (OPA) method. 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging activities assays were used to evaluate the antioxidant activity. Sonication pretreatment caused a higher degree of hydrolysis by chymotrypsin compared to high-pressure homogenization. Samples pretreated by high-pressure homogenization exhibited significantly (P<0.05) higher DPPH and ABTS radicals scavenging activity when hydrolyzed by trypsin and higher ABTS radical scavenging activity when hydrolyzed by chymotrypsin. The degree of hydrolysis increased with increasing hydrolysis time.The chymotrypsin was significantly (P<0.05) more effective than trypsin in the hydrolysis of protein. High-pressure homogenization pretreatment and trypsin hydrolysis were considered as the best method for producing yeast protein hydrolysate with DPPH (297.36 µMTE/mg protein) and ABTS (1189.02 µMTE/mg protein) radicals scavenging activities.