Ginkgonis Semen (GS) is the seed of Ginkgo biloba Linné and a valuable material for herbal medicines and functional foods in China, Japan and Korea. The main bio-compounds of GS are GA, GB and GC like the leaves. There are many studies for the analysis of ginkgolides in the leaves or leaves extract of G. biloba because the leaves extract is a valuable material in pharmaceutical industries. However, there is no efficient analytical method for the quality control of GS based on the quantitation of ginkgolides because of matrix effect induced by different chemical composition. So, there are no content criteria of GS in Pharmacopoeia of Korea, Japan and China until now. This study aimed to develop HPLC method using ginkgolides based on the quantitation of GA, GB and GC for the quality control of GS with the optimization of sample preparation to enhance the analytical sensitivity and reproducibility. At first, defatting process using petroleum ether and liquid–liquid extraction were applied for sample preparation to remove matrix effect. The HPLC-ELSD method was developed with the mobile phase of a 0.5% aqueous acetic acid and methanol–acetonitrile solution (1:1 ratio) under gradient conditions. GA, GB and GC contents in GS were different between Korea and China. The mean quantity of Korean samples was 4.85 ± 2.33 μg/g GA, 48.38 ± 5.10 μg/g GB, and 37.83 ± 7.64 μg/g GC. Those contents of Chinese samples were higher than Korean samples as 9.39 ± 2.51 μg/g GA, 123.59 ± 26.24 μg/g GB and 53.39 ± 4.97 μg/g GC. It indicated that the discrimination of GS between Korea and China could be achieved by marker compound contents. Furthermore, the geographical discrimination of GS between Korea and China was confirmed by PCA using the quantitative data of marker compounds. By statistical analysis, the calculated content criteria of GS by regression method were 2.35 μg/g of GA, 29.20 μg/g of GB, and 27.75 μg/g of GC, based on dry weight. Thus, our HPLC method shows potential toward the development of a universal quality control methodology to quantify GS quality and origin.