The decrease of petroleum reserves and the global increase in energy demand has resulted in the focus of research toward exploration of alternate fuels using biological renewable sources. Biodiesel is one of these renewable energy forms. Microalgae are considered one of the most promising sources for biodiesel production. However, the potential use of microalgae in this field still needs to be explored since only about twenty species has been studied from the approximately 30.000 known species. Thereby, screening work on new potentially lipid overproducer strains is actually an active field. An overview of potential applications of multi-parameter flow cytometry in development of a biodiesel production strategy using microalgae is detailed in this review. Rapid and simultaneous measurements of different physiological parameters indicating diversity in marine and freshwater microalgal communities, biomass quality such as biochemical composition and viability of individual cells, isolation of targeted cells and obtention of axenic cultures are applications of flow cytometry that enable monitoring and optimisation of production of lipids from microalgae.