Systemic delivery of amikacin is a widely adopted treatment modality for severe infections like sepsis. However, the current course of treatment requires repeated bolus doses of amikacin, prolonged hospitalization, and continuous therapeutic monitoring to manage the severe adverse effects. Amikacin has short half-life, which further challenges the delivery of sufficient systemic concentrations when administered by intravenous route. To solve this issue, novel delivery systems, amikacin liposomes (Ak-lip) were developed and evaluated for its antibacterial efficacy (agar plate diffusion and resazurin microtiter assay) and in vivo drug release in Sprague-Dawley rats. The Ak-lip were prepared by modified thin film hydration method and optimized based on particle size and Zeta potential. The zone of inhibition for Ak-lip and amikacin was found to be 22 mm and 26 mm against Staphylococcus aureus. The minimum inhibitory concentrations (MIC) of amikacin and Ak-lip against Staphylococcus aureus were found to be 3 µg/mL and 9 µg/mL, and for Pseudomonas aeruginosa were 0.6 µg/mL and 0.9 µg/mL respectively. The in vivo pharmacokinetic parameters were determined using Gastroplus™. A significant difference in the pharmacokinetic parameters (AUC, Cmax) was observed between amikacin and Ak-lip. The developed formulation showed good colloidal stability and sustained release profile up to 72 h which can reduce dosing frequency, minimize hospitalization and improve bactericidal activity at lower concentrations paving the path for improved therapeutic interventions in the treatment of sepsis.