Sustained-release formulations for ocular delivery are of increasing interest given their potential to significantly improve treatment efficacy and patient adherence. The objectives of this study were (i) to develop a sustained-release formulation of spironolactone (SPL) using a biodegradable and injectable polymer, hexyl-substituted poly-lactic acid (hexPLA) and (ii) to investigate the ocular biodistribution and tolerability of SPL and its metabolites in rats in vivo over 1 month following a single intravitreal injection (IVT inj). The concentrations of SPL and its two principal active metabolites, 7α-thiomethylspironolactone and canrenone (CAN), in the different ocular compartments were determined at different time points (3, 7, and 31 days after IVT inj) using a validated ultra-high-performance liquid chromatography-mass spectrometry method. Systemic exposure following a single IVT inj of 5% SPL-hexPLA formulation was evaluated by quantifying SPL and its metabolites in the plasma. Ocular tolerability of the formulation was evaluated using in vivo retinal imaging and histology. In vitro release studies revealed a sustained release of SPL from 5% SPL-hexPLA for up to 65 days. In vivo studies showed that SPL and its metabolites were detected in all ocular tissues at 3 and 7 days post-IVT inj. At 31 days post-IVT inj, SPL and CAN were mainly detected in the retina. These results also highlighted the clearance pathway of SPL and its metabolite involving the anterior and posterior routes in the first week (days 3 and 7), then mainly the posterior segment in the last week (day 31). This study showed that a single IVT inj of 5% SPL-hexPLA in rats enabled sustained delivery of therapeutic amounts of SPL for up to 1 month to the retina without systemic exposure. This formulation may be of interest for the local treatment of diseases involving overactivation of the mineralocorticoid receptor in the chorioretina such as chronic central serous chorioretinopathy.