Background: Cubebin is a naturally occurring lactone lignan isolated from the plant species Piper cubeba. It exhibits a range of biological activities, including anti-inflammatory, antimicrobial, and anticancer properties. This compound has garnered significant interest for its potential therapeutic applications in traditional and modern medicine. Aim: This study aimed to develop a novel stability-indicating High-Performance Thin Layer Chromatography (HPTLC) technique that is simple yet precise in estimating cubebin in crude drug and marketed formulations. Method: Cubebin was estimated using precoated TLC silica gel 60 F254 plate as a stationary phase and n-hexane: ethyl acetate: formic acid: methanol (6:3.5:0.5:0.3 v/v/v/v) as a mobile phase with the chamber saturation time of 20 mins. Key Findings: Cubebin was detected and quantified at the wavelength of 283nm. The Rf value of cubebin in standard solution, Piper cubeba extract, Dhanwantari Vati, and Khadiradi Vati was found to be 0.494±0.0015, 0.492±0.002, 0.490±0.0020, 0.499±0.001 respectively. The calibration curve exhibited linearity within the range of 3000-13000 ng per band with a correlation coefficient (r2) of Cubebin was 0.9968. The method developed was utilized for the quantitation of cubebin in Piper cubeba and both the marketed formulations. Conclusion: A novel HPTLC method was developed and validated in accordance to ICH guidelines for the analysis of cubebin in crude drug and marketed formulations. The method was found to be simple, precise, accurate, and specific. This method is suitable for quality control and stability testing of cubebin-containing formulations.