This study aimed at developing an efficient, fast and environmentally-friendly method to quantify neutral lipid contents in yeast. After optimising the fluorescence instrument parameters and influence of organic solvent concentrations, a new method to quantify neutral lipids in yeast based on fluorescence was demonstrated. Isopropanol and Nile red in concentrations of 5% (final volume%) and 500 μg/L, respectively, were added to washed cells suspended in potassium chloride phosphate buffered saline (PBSKCl). Fluorescence was measured after 10 min in the dark. Glyceryltrioleate was used as model lipid and the calibration curve showed linearity (R2 = 0.994) between 0.50 and 25 mg/L. Compared with traditional gravimetric analysis, the developed method is much faster and uses less organic solvents. Lipid contents determined by fluorescence and gravimetry were the same for some strains, but for other strains the lipid contents determined by fluorescence were less. This new method will therefore be suitable for fast screening purposes.