Abstract Lipids extracted from microalgae for biofuel production have drawn the attention of researchers. Various methods for quantification of neutral lipids, such as solvent extraction or gravimetric, require a large volume of sample and are time-consuming. In this work, Nile Red was employed to evaluate the lipid content in microalgae Scenedesmus sp. through 2D fluorescence spectroscopy using chemometric modeling with a selection of wavelength pairs reducing the variable numbers. Cultivation of microalgae for 10 days was performed. Samples were taken starting after the 5th day of cultivation. Microalgae samples were dried using an oven with tangential airflow and the conventional Bligh and Dyer method for lipid extraction was employed. Before the measurement of fluorescence, the samples were diluted in ethanol at 40°C for 10 minutes, and then Nile Red solution (10 µg/mL ethanol) was added. The results of lipid quantification using the proposed technique achieved mean absolute percentage error (MAPE) lower than 5% and correlation coefficients around 0.99 in the test phase. Linear models using four excitation-emission pairs of fluorescence were fitted and were selected based on Pure Spectral Chemometric Modelling (PSCM). The method for lipid quantification using predictive linear models and 2D fluorescence proved to be robust and fast (requiring less than 30 min) compared to the conventional gravimetric method (which takes at least 5 hours) to obtain comparable results.