A robust and reliable method has been developed for the measurement of β-glucan in mushroom and mycelial products. Total glucan (plus free glucose and glucose from sucrose) was measured using controlled acid hydrolysis with H2SO4 and the glucose released specifically was measured using glucose oxidase/peroxidase reagent. α-Glucan (starch/glycogen) plus free glucose and glucose from sucrose were specifically measured after hydrolysis of starch/glycogen to glucose with glucoamylase and sucrose to glucose plus fructose with invertase and the glucose specifically measured with GOPOD reagent. β-Glucan was determined by the difference. Several acid and enzyme-based methods for the hydrolysis of the β-glucan were compared, and the best option was the method using H2SO4. For most samples, similar β-glucan values were obtained with both the optimized HCl and H2SO4 PROCEDURES: However, in the case of certain samples, specifically Ganoderma lucidum and Poria cocus, the H2SO4 procedure resulted in significantly higher values. Hydrolysis with 2 N trifluoroacetic acid at 120°C was found to be much less effective than either of the other two acids evaluated. Assays based totally on enzymatic hydrolysis, in general, yielded much lower values than those obtained with the H2SO4 procedure.