Hypercholesterolemia is a main contributing risk factor to cardiovascular disease. Lipase inhibition lessens cholesterol biosynthesis in the liver. Statins are commonly used for hypercholesterolemia treatment. A lot of studies on natural products had been done to investigate potential inhibitors against lipase. Catechin as the essential compound in Uncaria gambir Roxb is conceivable for hypercholesterolemia treatment. This study aims to determine the inhibitory effect of extracts, ethyl acetate fraction, and catechin isolate of Uncaria gambir on lipase activity. Gambir leaf was extracted in water, the fractionation was caried out in ethyl acetate solvent while the isolation of catechin gambier was carried out in ethyl acetate-hexane as the solvent. Phytochemical analysis of extract and isolate was conducted by HPLC. The inhibitory activity of lipase enzymes was carried out using a lipase assay kit by ELISA as well as orlistat as the standard drug. This study has resulted in 68.32%, 92,26% and 96.74% catechin contained in extract, ethyl acetate fraction, and catechin isolate. The in-vitro inhibition assessment of lipase activity indicated that catechin isolate of Uncaria gambir was the most potential inhibitor of lipase (IC50 = 0.102 mg/mL), here in after ethyl acetate fraction supernatant (IC50 = 0.386 mg/mL), while extract and orlistat showed relatively less inhibition (IC50 = 0.680 mg/mL and IC50 = 0.645 mg/mL). Catechin isolate from Uncaria gambir showed strong inhibition of lipase activity. The outcome showed an equal correlation that the higher catechin, the higher inhibitory of lipase.