Soy isoflavones are generally analyzed in accordance with the official method of AOAC International (OMA) 2001.10 and OMA 2008.03. However, this method is time-consuming and requires a large amount of sample. Therefore, the development of a better analytical method for soy isoflavones is required. In this study, OMA 2001.10 and OMA 2008.03 were improved to develop a quantification method for soy isoflavones. As a result, we shortened the analysis time using HPLC. The isoflavone content of a soybean sample was measured according to the improved method in triplicate, and the measurements were repeated on three different days. Both the repeatability and intermediate precision (within laboratory) for the quantification of the isoflavone content (aglycone) of the soy sample were good. The recovery of isoflavones spiked into a soy sample at two different concentrations ranged from 94% to 105%. The multilaboratory validation study with 7 participating laboratories showed satisfactory interlaboratory precision; all HorRat values were <2.