Lipofuscin in the retinal pigment epithelium (RPE) is the major source of fundus autofluorescence (FAF). A technical challenge to accurately quantify the FAF intensities, thus the lipofuscin concentration, is to compensate the light attenuation of RPE melanin. We developed the VIS-OCT-FAF technology to accomplish optical coherence tomography (OCT) and FAF simultaneously with a single broadband visible light source. We demonstrated that light attenuation by RPE melanin can be assessed and corrected using the depth-resolved OCT signals. FAF images from albino and pigmented rats showed that without compensation, FAF signals from pigmented rats are lower than that from albinos. After compensation, however, FAF signals from pigmented rats are higher. This finding is supported by measurements of lipofuscin fluorophore A2E in the RPE using liquid chromatography/mass spectrometry (LC/MS) showing that compensated FAF intensities correlate linearly with A2E contents. The present work represents an important step toward accurately assessing RPE lipofuscin concentrations by FAF.