Apolipoprotein A-I (apoA-I), the main protein component of high density lipoprotein (HDL), is the principal facilitator of cholesterol efflux from cholesterol-laden macrophages. Lipid-free apoA-I is the preferred substrate over lipid-associated apoA-I for cholesterol mobilization by the membrane transporter ATP binding cassette A1, which is responsible for more than 60% of cellular cholesterol efflux from cholesterol-laden macrophages. However, more than 95% of apoA-I is lipid-bound and associated with plasma mature HDL. To study the mechanisms that promote the production of lipid-free/lipid-poor (cholesterol-efflux capable) apoA-I in the arterial walls, we developed an apoA-I variant capable of reporting the lipidation-state of apoA-I in real-time.We employed fluorescence resonance energy transfer (FRET) to generate an apoA-I reporter with lipidation-state specific fluorescence. ApoA-I's four endogenous tryptophans (Trp) were substituted with phenylalanines and a single Trp was substituted in at position 19, as the FRET donor. A cysteine residue substituted in at position 136 was labeled with the fluorophore AEDANS, as the FRET acceptor. The resultant apoA-I variant, apoA-I:W19-AED136, was lipidated to varying degrees producing rHDL of different sizes. The fluorescence emission spectrum of lipid free apoA-I:W19-AED136 and each of the rHDL particles was collected. Structural differences in the conformation of lipid-free apoA-I and apoA-I associated with different rHDL sizes altered the relative positions of the FRET donor-acceptor pair, leading to lipidation state specific fluorescence “fingerprints”. Lipid-free apoA-I:W19-AED136 showed the highest degree of energy transfer (E=0.571), and apoA-I exhibited decreasing levels of energy transfer with increasing rHDL particle size (7.8 nm (E=0.387), 8.4 nm (E=0.0780), and 9.6 nm (E=0.0334)).ApoA-I:W19-AED136 was successfully used to measure the transition rate of apoA-I between lipid-associated and lipid-free states, potentially, the rate limiting step of macrophage cholesterol efflux in the atherosclerotic plaque.