Mushrooms, an essential part of fungal biota, have been widely investigated due to their nutritional, prophylactic, and therapeutic benefits. The lipid-lowering pharmacological activity of medicinal mushrooms has garnered attention, which can be adjuvant to treat hypercholesterolemia. Polysaccharides extracted from some kinds of medicinal mushrooms, such as Lentinus edodes, Agaricus blazei, and Ganoderma Lucidum Karst, exhibit beneficial anticholesterolemic activities that promote the metabolism and excretion of cholesterol in the liver and reduce the accumulation of cholesterol in the body. When we need to determine the anticholesterolemic effect of medicinal mushrooms in the laboratory, enzyme reaction assay, high-performance liquid chromatography, and liquid chromatography-mass spectrometry are commonly employed as quantitative analysis methods in measuring the fat index like total cholesterol. This chapter summarizes the experimental principle, material list, method process, and a few notes on the partial measurement process in the anticholesterolemic effect of medicinal mushrooms.