This study investigated the pharmacokinetics of levofloxacin niosomes following a single dose administered intraperitoneally to Sprague Dawley rats, using conventional unentrapped drug as reference. Guided by results of previous studies, an experimental design software (DoE Fusion One) was used to design screening experiments that provided factor combinations and processing variable settings that resulted in an optimized formulation. Optimized niosomes of levofloxacin were prepared, characterized and evaluated in vitro for drug release rate. The well-characterized formulation was used for in vivo study in Sprague Dawley rats in a non-cross over parallel study. One of two groups of rats (n = 6 each) was injected intraperitoneally with conventional levofloxacin and the other with levofloxacin niosomes at 7.5mg/kg/dose. Blood samples were collected at ½, 1, 2, 4, 8, 12, 18, 24, 48 and 72 hours via tail snip. Drug concentrations were determined using a validated high-performance liquid chromatographic (HPLC) method. The plasma concentration versus time data from rats were fed into the Gastroplus software (Simulations Plus, CA) and used to model the pharmacokinetic profiles of the i.p. levofloxacin and its niosomes. The optimized drug-loaded niosomes used for in vivo rat study had a mean size of 368.8 (± 11.0 SE), encapsulation efficiency (EE) of 34.21 %, Zeta potential of -42.10 (± 2.07 SE) and mean polydispersity index (PDI) of 0.316 (± 0.014 SE). Following i.p. injection, levofloxacin release was controlled with less than 80 % released in 6 h and about 90% released in 12 h. Modeling of levofloxacin bio-disposition following i.p. injection in rats with Akaike information criterion (AIC) and Schwarz criterion (SC) indicated that the niosomes formulations are best fitted to one-compartment model while the conventional drug formulation followed two-compartment model. The niosomal formulation appeared to alter the biodistribution of levofloxacin, and concentrated the drug in the vascular compartment with slow, sustained distribution outside of the vascular compartment. Overall, the niosomes formulation showed significant increase in levofloxacin mean residence time (MRT), prolonged its elimination half-life (t1/2) and decrease its volume of distribution (Vd). Encapsulation of levofloxacin in niosomes altered its pharmacokinetic profile. It appears that, by vesicular encapsulation, niosomes altered levofloxacin biodistribution possibly by preventing its binding to plasma protein while correspondingly increasing its residence time in systemic circulation. Thus, by protecting intraperitoneally injected levofloxacin en route into the plasma, niosomes significantly enhance its delivery to, and concentration in, the intravascular compartment with enanced potential for treating intravascular conditions like bacteremia.