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Flow cytometry is the gold-standard laser-based technique to measure and analyze fluorescence levels of immunostaining and DNA content in individual cells. It provides a valuable tool to assess cells in the G0/G1, S, and G2/M phases, and those with polyploidy, which holds prognostic significance. Frozen section analysis is the standard intraoperative assessment for tumor margin evaluation and tumor resection. Here, we present flow cytometry as a promising technique for intraoperative tumor analysis in different pathologies, including brain tumors, leptomeningeal dissemination, breast cancer, head and neck cancer, pancreatic tumor, and hepatic cancer. Flow cytometry is a valuable tool that can provide substantial information on tumor analysis and, consequently, maximize cancer treatment and expedite patients’ survival.