A new, specific, sensitive, selective, precise, and reproducible high-performance thin-layer chromatographic (HPTLC) method has been established for study of the stability of 3-acetyl-11-keto-β-boswellic acid (AKBA). HPTLC was performed on aluminium foil plates coated with 200 μm silica gel 60F254. Linear ascending development with toluene-ethyl acetate 7:3 (v/v) was performed at room temperature (25 ± 2°C) in a twin-trough glass chamber saturated with mobile phase vapour. Compact bands (RF 0.52 ± 0.02) were obtained for AKBA. Spectrodensitometric scanning was performed in absorbance mode at 250 nm. Linear regression analysis of the calibration plots showed there was a good linear relationship (r2 = 0.9989 ± 0.0002) between peak area and concentration in the range 200–1200 ng band−1. The method was validated for precision, recovery, robustness, specificity, and detection and quantification limits, in accordance with ICH guidelines. The limits of detection and quantification were 3.06 and 9.29 ng band−1, respectively. The recovery of the method was 99.35–100.21%. AKBA was subjected to various stress test conditions — acid and alkali hydrolysis, oxidation, photodegradation, and dry and wet heat treatment. Degradation products were well resolved from the pure drug with significantly different RF values. Statistical analysis showed the method could be successfully applied for the estimation of AKBA in herbal extract and in nanoparticles. Because the method could effectively separate the drug from its degradation products, it can be regarded as stability-indicating.