This study aims to isolate, purify, and partially characterize protease inhibitors (PIs) from chickpea (Cicer arietinum) seeds using sequential protein purification techniques. Crude protein extract was prepared from defatted chickpea seed flour. The extract was subjected to ammonium sulfate fractionation, followed by ion exchange chromatography (DEAE-Cellulose) and gel f iltration chromatography (Sephadex G-100). Purity and molecular weight were assessed by SDS-PAGE. Inhibitory activity against trypsin was measured spectrophotometrically using BAPNA as substrate. A purification fold of approximately 28.6 with a specific activity of 320 U/mg was achieved. SDS-PAGE revealed a single band corresponding to a molecular weight of ~18 kDa. The purified inhibitor showed stability over a pH range of 5–9 and temperatures up to 60°C. A simple, efficient two-step chromatography protocol successfully purified a potent, low-molecular-weight trypsin inhibitor from chickpea seeds, which may have applications in nutrition, plant defense studies, and therapeutic development.