Reduction of low-density lipoprotein cholesterol by statin therapy has only modestly decreased coronary heart disease (CHD)-associated mortality in developed countries, which has prompted the search for alternative therapeutic strategies for CHD. Epidemiologic and interventional studies have clearly established an inverse association between plasma levels of high-density lipoprotein (HDL) cholesterol and incidence of atherosclerosis. The atheroprotective benefits of HDL are not only dependent on HDL concentrations (quantity), but also on HDL function (quality). Therefore, several techniques have been recently developed to assess the different properties of HDL. Because reverse cholesterol transport (RCT) is considered a key player in the beneficial action of HDL, this review focuses on the different methods used to evaluate cholesterol efflux. Measuring the in vivo function of HDL could be of significant importance for both the clinical evaluation of an individual patient and to evaluate the effectiveness of different RCT-enhancing therapeutic approaches.