An automated flow cytometric (FCM) detection method has been developed and validated with a simple diagnostic procedure in parasitized erythrocytes of Plasmodium berghei-infected rats using the nucleic acid-binding fluorescent dye YOYO-1. High levels of reticulocytes were detected during the course of the infection, ranging from 1·2–51·2%, but any RNA potentially confounding the assay could be removed by digestion with RNAse. The cell counts of uninfected, infected, and nucleated cells occurred with high precision. The cells were divided into different populations according to their physical or chemical properties but various factors within the assay such as cell fixation, RNA digestion, and compensation required optimization. In this study, FCM greatly simplified and accelerated parasite detection, with a mean precision of 4·4%, specificity of 98·9% and accuracy of 101·3%. The detection and quantitation limits in the assay were 0·024% and 0·074% parasitaemia, respectively. Overall, the parasite counts by FCM measurement correlated highly (r2=0·954–0·988) with the parasitaemia measured by light microscopical analysis when animals treated with suppressive, clearance, and curative doses of novel antimalarial drugs were examined. The lower levels of parasitaemia (30%) detected by microscopy compared to FCM may be related to a number of schizonts externally attached to the erythrocyte membranes that normally would not be included in microscopy counting. Lower sampling error and reliable identification of rodent erythrocyte parasites based on the principles of FCM have replaced the traditional blood smear in our laboratory.