Published protocols for sugar extraction from soybean seed are time consuming and difficult to use in large-scale breeding selections. A rapid and reproducible procedure was established in this research to analyze soluble sugar content in soybean seed. Seed samples of 15 g were ground with a regular coffee grinder and screened through a 250 µm standard testing sieve. Soluble sugars were extracted from 0.15 g soybean flour with 1.5 ml distilled water on a horizontal shaker at 250 rpm for 20 min. Supernatants of 500 µl were purified with 700 µl acetonitrile for 30 minutes, dried on a hot bath incubator, and re-dissolved in 200 µl 65% acetonitrile. Major sugars including glucose, fructose, sucrose, raffinose, and stachyose were separated and quantified with a Dionex DX500 HPLC system equipped with a GS50 pump, ED40 electrochemical detector, and a CarboPac PA10 pellicular anion-exchange resin column. Technical details of sugar extraction and quantification will be presented.