Purpose: Pentobarbital is a sedative agent to limit children motion during computed tomography or magnetic resonance imaging (MRI) and ensures the successful completion of the imaging procedure. However, data on rectal drug formulation and its stability in practice are not available. The aim of this study was to formulate and evaluate the stability of a ready-to-use rectal pentobarbital gel. Methods: The formulation consisted of a hydrated gel containing 25 mg/mL of pentobarbital sodium, packaged in 10-mL amber glass bottles and stored at either 22°C to 25°C or 2°C to 8°C. At each predetermined time point, samples were taken for visual inspection, pH measurement, and analysis by a validated stability-indicating high-performance liquid chromatography (HPLC) method. The viscosity parameters of the hydrogel formulation were assessed. Results: The freshly prepared rectal formulations appeared clear, colorless, and particular-free with pH readings of 9.75 to 9.83. Over the 90 days of the study period, there was no significant change in appearance or pH values for all stability samples. The HPLC results confirmed the chemical stability when stored at 2°C to 8°C or at 22°C to 25°C. Conclusion: Pentobarbital hydrogel 25 mg/mL are stable chemically at least 90 days and can be administered to children for an effective and fast sedation.