BACKGROUND Momordica charantia Linn. (Cucurbitaceae) fruits are well known for their beneficial effects in diabetes that are often attributed to its bioactive component charantin. OBJECTIVE The aim of the present study is to develop and optimize an efficient protocol for the extraction of charantin from M. charantia fruits. METHODS Response surface methodology (RSM) was used for the optimization of ultrasound-assisted extraction (UAE) conditions. RSM was based on a three-level, three-variable Box-Behnken design (BBD), and the studied variables included solid to solvent ratio, extraction temperature, and extraction time. RESULTS The optimal conditions predicted by the BBD were: UAE with methanol: Water (80:20, v/v) at 46°C for 120 min with solid to solvent ratio of 1:26 w/v, under which the yield of charantin was 3.18 mg/g. Confirmation trials under slightly adjusted conditions yielded 3.12 ± 0.14 mg/g of charantin on dry weight basis of fruits. The result of UAE was also compared with Soxhlet extraction method and UAE was found 2.74-fold more efficient than the Soxhlet extraction for extracting charantin. CONCLUSIONS A facile UAE protocol for a high extraction yield of charantin was developed and validated.