Frozen section studies are a useful method to rapidly define tumor malignancy and identify the extent of surgical resection. However, diagnosis with a frozen section is qualitative and sometimes difficult. Therefore a quantitative method for grading tumors is desired. We have already reported a technique of intraoperative flow cytometry (iFC) that supports intraoperative histopathological examination of frozen sections. In this study, we report an advanced system named 'Fully Automatic Rapid DNA Ploidy Analyzer' with a tissue pretreatment function and a freeze-dried reagent kit for cell staining. To evaluate our system, we analyzed samples from glioma patients who underwent open surgery for brain tumors. We observed obvious difference of the Malignancy Index (MI) between neoplastic and perilesional brain tissue (26.0 ±22.1% and 4.1 ±2.5%, respectively, P<0.001). Cut-off level for identification of the tumor in the biopsy specimen was 6.8% which provided 86% sensitivity and 81% specificity. We also obtained a good correlation between the MI and histological grade (WHO grading). Our new system also enabled finishing the process from sample preparation to the end of analysis in ten minutes or less. These results demonstrate that our fully automatic rapid DNA ploidy analyzer is feasible for rapid determination of glioma presence in a surgical biopsy sample.