This study aimed to develop an optimized depot injectable atorvastatin (ATR) biodegradable in situ gel (ISG) system with minimum initial burst using a central composite design. The factors selected were poly ( d, l-lactide-co-glycolide) (PLGA) concentration (X 1), molecular weight of polyethylene glycol (PEG) (X 2), and PEG concentration (X 3). The independent variables were the initial burst of ATR after 2 (Y 1) and 24 hours (Y 2). The optimized formulation was investigated using scanning electron microscopy, Fourier transform infrared spectroscopy, and in vitro drug release in phosphate-buffered saline of pH 7.4 for 72 hours. The in vivo pharmacokinetic study of the optimized ATR-ISG and the corresponding PEG-free ATR-ISG were conducted by intramuscular injection of a single dose (2 mg/kg) of ATR in male New Zealand White rabbits. A double-blind, randomized, parallel design was used in comparison with those of the marketed ATR tablet. Statistical analysis revealed that PLGA concentration and the molecular weight of PEG have pronounced effects on both Y 1 and Y 2. The optimized formulation was composed of 36.10% PLGA, PEG 6000, and 15.69% PEG, and exhibited characteristic in vitro release pattern with minimal initial burst. Incorporation of PEG in the formulation causes a slight decrease in the glass transition temperature value of PLGA, leading to a slight change in Fourier transform infrared spectroscopy spectrum due to possible interaction. Moreover, scanning electron microscopy photomicrograph showed smooth surface with disappearance of the cracks which characterize the surface of PEG-free formulation. The pharmacokinetic data for the optimized depot injectable ATR-ISG showed a significant ( P<0.05) decrease in maximum plasma concentration from 547.62 to 346.84 ng/mL, and increasing time to reach the maximum plasma concentration from 12 to 72 hours in comparison with the marketed tablet. The optimized ATR-ISG formulation has shown minimal initial drug burst which confirms the suitability of the ISG system in the prolongation of drug release in patients with chronic long-term therapy.