The present work aimed to formulate and evaluate Besifloxacin Hydrochloride (HCl) ophthalmic in situ gel for conjunctivitis, based on temperature triggered gelation system to increase ocular residence time, bioavailability and patient compliance. Different formulations were prepared by using Poloxamer 407 and Poloxamer 188 as temperature sensitive as well as mucoadhesive polymers. Design expert software version 11 was used to optimized the formula. Optimized batch having Poloxamer 407 (24.80% w/v) and Poloxamer 188 (14.53% w/v) was evaluated for visual appearance, clarity, gelation temperature, gelling capacity, rheological property, ex vivo, in vitro release and accelerated stability studies. Histopathological study was carried out using Goat cornea and revealed no change in histopathology of the tissues. Formulation was found to be effective against the Escherichia coli and Staphylococcus aureus. Cumulative % drug release of Besifloxacin HCl ophthalmic in situ gel after 8 hours was found to be 96.47 %.