Background: Diabetes mellitus become one of the biggest global health problems of the 21st century. Type 2 diabetes play role for the majority of cases of diabetes worldwide which is characterized by the increase of postprandial blood glucose level. Maintaining postprandial glucose level through inhibition of α-glucosidase is one of the essential strategies in the treatment of diabetes. Inhibitory effect of α-glucosidase was commonly used to identify active compounds potentially to treat diabetes. Natural resources have potency as antidiabetic that can be used in diabetes treatment. Objective: The objective of the study is to separate active fraction in the crude extract of Garcinia hombroniana leaves to facilitate obtaining a pure biologically active compound as the α-glucosidase inhibitor. Methods: Fractionation to separate active fraction was performed using column and thin layer chromatography methods while α-glucosidase inhibitory activity assay was performed in vitro using spectrophotometric methods at λ 400 nm. Results: Ethyl acetate and methanol extract of G. hombroniana yielded 14 and 12 fractions, respectively. Two fractions with the higher percent inhibition compared to other factions are fraction 8 from ethyl acetate extract (FEA8) and fraction 3 from methanol extract (FM3). The IC50 values of FEA8, FM3 and acarbose are 16.370 μg/mL, 59.042 μg/mL, and 39.534 μg/mL respectively. Conclusion: Fraction 8 from ethyl acetate extract of G. hombroniana leaves (FEA8) was separated and known in this study as the most bioactive α-glucosidase inhibitor agent compared with another extract, fractions, and acarbose.