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Cholesterol is an essential component of eukaryotic cellular membranes. Information about its subcellular localization and transport pathways inside cells are key for the understanding and treatment of cholesterol-related diseases. In this review we give an overview over the most commonly used methods that contributed to our current understanding of subcellular cholesterol localization and transport routes. First, we discuss methods that provide insights into cholesterol metabolism based on readouts of downstream effects such as esterif