Obesity, as a worldwide disease, have surged insurmountably and cause huge damage to humans - receding both life expectancy and quality of life. The inhibition of lipase is the most explored strategy for the treatment of obesity until now. However, there is few kinds of anti-lipase products in the market and the commonly used drug - orlistat has many side effects. Natural products from medicinal herbs with anti-lipase activity may have potential for the treatment of obesity. In this study, high performance thin layer chromatography (HPTLC) combined with bioautography methods were used to simultaneously analyze steroidal saponins and homoisoflavonoids and screen potential lipase inhibitors in Ophiopogonis Radix. Five anti-lipase compounds including ophiopogonin D, ophiopojaponin C, ophiopogonin D′, ophiopogonin C′ and methylophiopogonanone B were rapidly screened. Among them, ophiopojaponin C showed the best inhibitory effect at the same concentration. Compared to the samples from Sichuan and 3-year-old samples from Zhejiang, the total lipase inhibition activities of 1 or 2-year-old Ophiopogonis Radix from Zhejiang seemed to be stronger, which might be a suggestion for the selection of suitable Ophiopogonis Radix samples in the clinical application. This study was supported by the National Natural Science Foundation of China (82104354), Guangdong Basic and Applied Basic Research Foundation (2022A1515012154), the funding grant from University of Macau (MYRG2022-00198-ICMS), and the Science and Technology Development Fund, Macau SAR (0070/2021/AGJ).