Intracellular lipid homeostasis is regulated by multiple mechanisms devoted to the tight control of cholesterol levels. Cholesterol efflux to extracellular acceptors represents a cellular response to excess accumulation of lipids that occurs by both passive and active processes and was shown to exert a beneficial, antiatherosclerotic activity. Up to now 3 lipid transporters responsible for cholesterol removal from cells have been characterized: ATP Binding Cassette A1 (ABCA1), ATP Binding Cassette G1 (ABCG1) and Scavenger Receptor Class B Type I (SR-BI). These proteins widely differ in the pathway of efflux they mediate, as well as in the nature of extracellular acceptors they interact with. The experimental investigation of cholesterol efflux pathways can be efficiently performed in vitro, following precise criteria in the selection of cell types and extracellular acceptors to specifically investigate the mechanism involved. The aim of this review is to describe how lipid transporter-mediated cholesterol efflux influences the intracellular trafficking of cholesterol and to provide methodological considerations for experimental evaluation of this process.