Brassica napus (L. cv Jet Neuf) microspore-derived (BnaMD) cell suspension cultures were used to study the biochemistry and molecular biology of plant lipid metabolism. The cell suspension cultures may also be a potentially useful trait-testing tool for further improvement of oilseed rape, the world's third largest vegetable oil crop. Nile red fluorescence methods have been used to assess neutral lipid accumulation in microorganisms and mammalian oocytes but have not been established for plant cell suspension cultures. In this study, a Nile red method was established and optimized for triacylglycerol (TAG) content analysis in BnaMD cell suspension cultures. Nile red fluorescence was easily determined in 96-well plates using a 490 nm excitation wavelength and a 595 nm emission wavelength without any significant interference from other cellular components. The lipid content of the cell suspension cultures at different growth stages was determined using a conventional lipid analysis method and the Nile red fluorescence method. There was a strong positive correlation between the Nile red fluorescence and TAG content in the cell suspension cultures. Therefore, the TAG content of BnaMD cell suspension cultures can be rapidly and robustly determined using a Nile red fluorescence-based method that can potentially be adapted for application to other plant cell suspension cultures.