Fluorescent dyes offer a useful method for the measurement of intracellular lipids. They are inexpensive and require simple optical measurement instrumentation, whilst simultaneously providing high throughput application. Nile Red is a hydrophobic, metachromatic dye which has been widely used for detection of intracellular lipids. However, Nile Red fluorescence depends on its concentration, microenvironment polarity, incubation time and, therefore, requires strain specific optimization. Hence, neutral lipids in Chlorella emersonii and Pseudokirchneriella subcapitata cannot be quantified using existing Nile Red methods developed for other microalgae strains and, therefore an optimised procedure for these strains is required. In this method development, the optimal excitation and emission wavelengths were selected based on the solvent used for Nile Red dissolution. The effect of Nile Red concentration, microalgae cell concentration, incubation time on fluorescence intensity was explored and optimised. Quintuplet assay repeats were executed for increased assay robustness for two microalgae strains, Chlorella emersonii and Pseudokirchneriella subcapitata, with protocol reliability confirmed by fluorescence microscopy. In brief, 20% (v/v) DMSO containing 10μg/ml and 5μg/ml Nile red was found to be ideal concentration for neutral lipid estimation in Chlorella emersonii and Pseudokirchneriella subcapitata respectively when an incubation time of 60mins and 40mins at 40°C was used. This optimised Nile Red protocol is a robust, simple and cost-effective method for neutral lipid quantification in Chlorella emersonii and Pseudokirchneriella subcapitata.