Orbifloxacin (ORBI) was subjected to different ICH (Q1A(R2)) prescribed stress conditions of thermal stress, hydrolysis, oxidation and photolysis. It was stable to dry heat (60 ºC) and photolysis (UV-VIS) in solid form. It showed extensive decomposition under hydrolytic and photolytic conditions in acid, base and neutral solutions. Degradation was also observed in oxidative condition. Degradation products of ORBI formed under different forced conditions were characterized through LC–MS studies. In total, eleven major degradation products (DP1-DP11) were detected. Successful separation of drug and degradation products formed under various stress conditions was achieved on a analytical column SymmetryShield Waters RP18, 5 µm, 250 x 4.6 mm using 5% acetic acid and methanol as mobile phase in a gradient mode at flow rate of 0.5 ml/min. The peaks were detected using a PDA detector set at 290 nm. The method was extended to LC–APCI-MS for characterization of the degradation products and the pathways of decomposition were proposed.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Estadual Paulista Júlio de Mesquita Filho, Departamento de Farmacos e Médicamentos, Faculdade de Ciências Farmacêuticas de Araraquara, Araraquara, Rodovia Araraquara-Jaú, km1, Campus, CEP 14801-902, SP, BrasilSchool of Pharmacy and Medical Sciences, University of South Australia, Adelaide, AustraliaUniversidade Estadual Paulista Júlio de Mesquita Filho, Departamento de Farmacos e Médicamentos, Faculdade de Ciências Farmacêuticas de Araraquara, Araraquara, Rodovia Araraquara-Jaú, km1, Campus, CEP 14801-902, SP, Brasil