The intracellular concentration of internal nitrogen (the “cell nitrogen quota”) is crucial to explain the rate at which phytoplankton populations grow. Hence, understanding changes in cell nitrogen quota is informative on aquatic primary productivity, phytoplankton ecology, eutrophication, and algal blooms. However, current methods to directly monitor per-cell nitrogen quota remain inaccurate, expensive, and time consuming. This study tested the hypothesis that nitrogen limitation triggers systematic optical changes in single cells, which can be rapidly and accurately monitored with a standard flow cytometer. The freshwater microalgae Desmodesmus armatus, Mesotaenium sp., Scenedesmus obliquus, and Tetraëdron sp. were reared in nitrogen-limited batch culture conditions across two treatments of initial population densities and monitored for cell nitrogen quota, medium nitrogen, and optical flow cytometric properties of red fluorescence and forward and side light scatters. Changes in nitrogen quota could be described with high accuracy (R2 = 0.9) from observations of flow cytometric variables and medium nitrogen, and the relationship did not change across different species or initial population sizes. Red fluorescence was the most important variable explaining 77 % of the total variability in total cell nitrogen and up to 87 % when combined with side light scatter, the second most important variable. Our results indicate that optical flow cytometric variables are a convenient and reliable method to estimate nitrogen quota in microalgal cells.