Investigation of yeast neutral lipid accumulation is important for biotechnology and also for modelling aberrant lipid metabolism in human disease. The Nile red (NR) method has been extensively utilised to determine lipid phenotypes of yeast cells via microscopic means. NR assays have been used to differentiate lipid accumulation and relative amounts of lipid in oleaginous species but have not been thoroughly validated for phenotype determination arising from genetic modification. A modified NR assay, first described by Sitepu et al. (J Microbiol Methods 91:321-328, 2012), was able to detect neutral lipid changes in Saccharomyces cerevisiae deletion mutants with sensitivity similar to more advanced methodology. We have also be able to, for the first time, successfully apply the NR assay to the well characterised fission yeast Schizosaccharomyces pombe, an increasingly important organism in biotechnology. The described NR fluorescence assay is suitable for increased throughput and rapid screening of genetically modified strains in both the biotechnology industry and for modelling ectopic lipid production for a variety of human diseases. This ultimately negates the need for labour intensive and time consuming lipid analyses of samples that may not yield a desirable lipid phenotype, whilst genetic modifications impacting significantly on the cellular lipid phenotype can be further promoted for more in depth analyses. 