In the wake of global warming and rapid fossil fuel depletion, microalgae emerge as promising feedstocks for sustainable biofuel production. Nile red staining acts as a rapid diagnostic tool to measure the amount of biodiesel-convertible lipid that the cells accumulate. There is a need for the development of a more uniform staining procedure. In its first phase, this study examined the dependence of microalgal Nile red fluorescence (Tetraselmis suecica) in terms of its most pertinent staining variables. A quadratic surface model that successfully described the Nile red fluorescence intensity as a composite function of its variables was generated (..2= 0.86). Cell concentration was shown to have a significant effect on the fluorescence intensity. Up to a certain threshold, fluorescence intensity was shown to increase with Nile red dye concentration. In its second phase, the study reviewed findings from previous Nile red studies to elucidate some of the fundamental mechanism underlying the diffusion of Nile red dye molecules into the microalgal cells and their subsequent interaction with intracellular lipids.Through the review process, we were able to develop a simple framework that provided a set of guidelines for the standardization of the Nile red staining procedure across different microalgal species.