Abstract: Objective: This study aimed to investigate angiotensin converting enzyme (ACE) inhibitory activity derived from Indonesian native buffalo meat and its purified protein using SEP-PAK Plus C18. Methodology: Buffalo meat was hydrolyzed with pepsin, trypsin and a combination of pepsin and trypsin. These hydrolysates were assessed for dissolved protein, the molecular weight of proteins and ACE inhibitory activity. The hydrolysate with the highest ACE inhibitory activity was subsequently purified using SEP-PAK Plus C18. The molecular weight of the proteins was analyzed using descriptive statistics and the dissolved protein content and ACE inhibitory activity were evaluated in a complete randomized design. Results: Buffalo meat contained protein (22.66±0.2%) and dissolved protein (3.67±0.59 mg mL–1). Enzymatic hydrolysis significantly elevated the level of dissolved protein and decreased the molecular weight of the proteins. The dissolved protein content following hydrolysis by pepsin, trypsin and a combination of pepsin and trypsin was 10.91±0.4, 8.09±0.8 and 10.90±0.4 mg mL–1, respectively. The molecular weight of proteins in the buffalo meat homogenate ranged from 40-70 kDa, whereas, the hydrolysates were generally less than 40 kDa. The ACE inhibitory activity of the purified hydrolysate (IC50 = 75.8±20 μg mL–1) was approximately twice that of the hydrolysate (IC50 = 133±18 μg mL–1). Purification using SEP-PAK Plus C18 increased ACE inhibitory activity. Conclusion: Buffalo meat hydrolyzed by pepsin had the highest ACE inhibitory activity compared with other hydrolysates. Purification using SEP-PAK Plus C18 almost doubled the ACE inhibitory activity.