Adipogenesis of preadipocytes in culture has been frequently used to study the molecular basis and effect of drugs on fat cell conversion. However, after adipogenic induction, cells respond to the inducing agent with various speeds of conversion and fat accumulation, which complicates direct molecular and biochemical analyses. Here we present a simple and sensitive method to detect and quantify fat accumulation inside cells by flow cytometry. Using this method, we detected elevated levels of cytoplasmic granularity that correlated well with an increased level of fat accumulated inside cells after adipogenic conversion. We further demonstrated the ability of this method to monitor and quantify fat cell maturation within a complex population of cells and to identify and collect the fat cells with similar fat storage for further analysis. Flow cytometry offers distinct advantages over existing detection systems for cytoplasmic lipid staining and lipid extraction and could represent a powerful analytical tool to monitor the effect of chemicals and biological molecules on fat cell conversion and maturation. Moreover, in combination with a cell sorting facility, our method offers a simple and efficient means of collecting fat cells of specific status for further analysis.