The widely applied Nile Red (NR) method allows near real-time monitoring of microalgal neutral lipid accumulation. When added to a culture sample, optimally, the fluorescent dye NR penetrates the microalgal cell wall staining the intracellular neutral lipids, and the measured fluorescence is linearly correlated to the neutral lipid concentration. Here I describe an optimization protocol for determining the optimal staining parameters for each new microalgal species, followed by a basic NR staining protocol to be applied for monitoring of microalgal neutral lipid accumulation.