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Three new process-related impurities were detected in levofloxacin sample by reversed-phase liquid chromatographic analysis method described in USP. Based on chromatographic elution order impurities were marked as Imp-1, Imp-2 and Imp-3. Impurities were isolated from Mother Liquor (ML) collected during various stages by using flash chromatographic method. These impurities were well characterized by Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) and Nuclear Magnetic Resonance (NMR). Mechanism of their formation is also discussed in this communication.