Extraction and quantification of soluble sugars in soybean are important for breeders in developing new lines with desirable amount of sugars. Extraction solvent, temperature, time, and solvent-to-sample ratio were studied to identify the optimal conditions for extraction of soluble sugars from non-defatted soybean meal. The sugar appropriate for preparing the standard curve in the phenol-sulfuric acid method for sugar quantification in soybean extract was also evaluated. Water and 10, 50 and 80% (v/v) ethanol were used as the solvents. Extraction temperatures of 25, 50, and 80oC, for 15, 30 and 60 min and solvent-to-sample ratios of 5:1, 10:1 and 15:1 (v/w) were combined with different solvents. Extractions from sample sizes of 0.1 g and 1 g were also evaluated. Raffinose was shown to be the most appropriate sugar in the preparation of the standard curve in the phenol-sulfuric acid method for an accurate estimation of the soluble sugars in soybean. Among the 47 combinations of extraction conditions, the majority produced similar amounts of sugars. Solvent and the interaction between solvent and temperature were found to be the most important factors in the extraction of soybean sugars. Interactions between temperature and solvent-to-sample ratio were also important. Although 10% and 50% ethanol were as effective as water under various extraction conditions, ethanolic extractions were more laborious and their extracts required further cleaning to remove lipids for HPLC analysis. The recommended extraction protocol thus included the use of water as the solvent at a solvent-to-sample ratio of 5:1 at 25oC or 50oC for 15 min. In this study, 80% ethanol was not as effective as all the other solvents. The extraction with 0.1 g sample showed a similar result as the extraction with 1 g sample indicating that accurate quantification of total sugar in soybean could be achieved with small samples.