An assay based on Nile red fluorescence was developed for quantitative analysis of triglycerides, a common cellular component with important biological functions and is routinely analyzed for diagnosis of metabolic disorders and as an important feedstock of food industry and biodiesel production. Based on studies on the Nile red fluorescence of pure, binary, and ternary systems of triglycerides, ethanol, and water, 20% ethanol aqueous solution was determined to be the most suitable solvent for lipid fluorescence measurement. Excellent linearity was established for lipid samples in the range of 0.1- 0.5 mg/ml with several different lipid standards and vegetable oils. Results also suggest that the fluorescence of triglycerides was not sensitive to the fatty acid composition of lipids. This finding is important since it implies that the assay could potentially be used for the measurement of triglyceride content of different oil crops without causing significant variations. The results of this method were then verified by comparing with the results of the conventional gravimetric methods. The results of the fluorescence assay were consistently lower than that of the gravimetric method by approximately 10%. This phenomenon was tentatively attributed to the fact that the gravimetric method measures the total amount of lipophilic materials in samples while the fluorescence assay is selective to glycerides. Attempts were also made to apply this assay to estimate the lipid content of green alga Neochloris oleoabundans. However, the results were less than ideal due to the existence of interfering components in the extract of microalga samples that could significantly repress the fluorescence of lipids.