Niosomes are non ionic surfactant vesicles and potential surrogate for liposomes. The aim of the present investigation was to formulate and evaluate niosomes. The formulated ofloxacin niosomes were evaluated for their particle size, zeta potential, surface morphology, entrapment efficiency, in vitro drug release and in vivo pharmacokinetic studies. Niosomes of ofloxacin were prepared by thin film hydration technique using rotary flash evaporator. The formulated ofloxacin niosomes showed a vesicle size of 3.0-3.8 μm and zeta potential of -9 to -13 mV. The in vitro release studies showed 98.79% of ofloxacin release in sustained manner following first order kinetics. The stability study confirmed the stability of Ofloxacin niosomes. Pharmacokinetics studies of ofloxacin niosomes made with Span 60 showed increased C<inf>max</inf> AUC, AUMC, t <inf>1/2</inf> and MRT values compared to marketed intravenous ofloxacin product. The designed ofloxacin niosomes with span 60 showed good physicochemical properties, good stability, improved pharmacokinetic parameters, prolonged action and improved bioavailability than the commercially available conventional dosage form which might be a potential carrier system to improve the patient compliance and reduce the side effects. © 2014 Elsevier B.V., All rights reserved.; MEDLINE® is the source for the MeSH terms of this document.