To allow the comprehensive assessments of yeast expression systems, a simple and immediate method for simultaneously evaluating the expression level and plasmid maintenance in yeast was demonstrated. This method uses green fluorescent protein (GFP) and flow cytometry (FCM) and is characterized by a dual analysis of the average intensity of GFP fluorescence and the population of GFP-expressing cells. The FCM analysis of GFP fluorescence intensity rapidly quantifies the expression level without complex manipulations, such as the enzymatic reaction of a lacZ reporter assay. Moreover, the single-cell analysis revealed that the proportion of cells expressing GFP in the cell cluster reflects the plasmid retention rate; therefore, the FCM analysis of the GFP-expressing population allows the immediate estimation of the plasmid retention rate without the 2- or 3-day incubation required for colony counting. We show that the FCM analysis with GFP reporter is a suitable method to explore the hopeful expression vector and host strain or establish the several expression systems exhibiting the characteristic properties in yeast.