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Adulteration of synthetic pharmaceutical drugs in jamu is prohibited by law in Indonesia. Glibenclamide is one of the drugs commonly added to jamu. The purpose of this study was to obtain a valid high performance liquid chromatography (HPLC) method for qualitative and quantitative analysis of glibenclamide in antidiabetic jamu. HPLC separation was carried out with a Kromasil 100 C18 column (150 x 4.6 mm i.d, 5 μm particle size) using methanol: water (75:25) v/v as the mobile phase at a flow rate of 0.5 mL/min, UV detection was set at 301 nm. There was no potential interference from other compounds at the glibenclamide retention time (retention time of 5.10 minutes). The method has good linearity (r = 0.9936) in the range 10-50 mcg/mL. The detection limit of the method was 6.21 mcg/mL while the quantitation limit was 20,71 mcg/mL. The relative standard deviation (RSD) of intraday precision was 1.923%. The average recovery using standard addition method was 100.65 ± 16.89%. The application of the HPLC method for the analysis of five antidiabetic jamu samples obtained from the market in Purwokerto showed that glibenclamide was detected in one sample with a level of 1.88 ± 0.25 mcg/g.