The trend towards a healthier diet and lifestyle has expanded the functional mushroom market and continues to grow exponentially. Finished products now come in a variety of forms and flavours. However, the boom of the medicinal mushroom industry has led to an urgent need for the development of fit-for-purpose methods to support authenticity claims and improve consumer trust.    The key active component found in mushroom products is 1,3/1,6-β-glucan. While the measurement of 1,3:1,4-β-glucan found in cereals and the 1,3/1,6-β-glucan found in yeast can be achieved using a selective enzymatic hydrolysis approach, an equivalent method is not available for the measurement of 1,3/1,6-β-glucan in mushrooms, due to the complex structure presented. Instead, an indirect method involving subtraction of enzymatically determined a-glucan content from an acid hydrolysis/chemo-enzymatic derived “total glucan” measurement, can be employed as described by McCleary et al. in 2016. Although this method readily allows for the measurement of β-glucan in mushroom products which are contaminated with α-glucans comprising starch, maltodextrins and/or sucrose, other components may present challenges. As such, a number of assay parameters have been thoroughly investigated, and the results of this study are described herein.