Maistero-2 is a novel, non-toxic cholesterol-binding protein derived from an edible mushroom Grifola frondosa mRNA. Maistero-2 specifically binds to lipid membranes containing 3-hydroxy sterols with a lower cholesterol concentration threshold than cholesterol-binding domain 4 (D4) of perfringolysin O (PFO) and anthrolysin O (ALO). Maistero-2 binding is particularly sensitive to the size and conformation of the A-, B-, and D-ring of sterols but not very sensitive to modifications of the isooctyl side chain commonly found in phytosterols. EGFP-maistero-2 has been demonstrated to be a suitable tool to visualize the increase of cell surface cholesterol during the differentiation of Neuro 2a cells and heterogeneous cholesterol distribution between CD63-positive and LAMP1-positive late endosomes/lysosomes in HeLa cells. This chapter describes the detailed protocols for examining the binding of maistero-2 to lipids and model membranes and the labeling of cell surface and intracellular cholesterol by EGFP-maistero-2.