Hibiscus sabdariffa L. is famous for its medicinal properties. The in-vitro anti-inflammatory activity of the total ethanolic extract and different fractions of H. sabdariffa leaves was evaluated. All samples demonstrated anti-inflammatory effect against the pro-inflammatory mediators, TNF-α, IL-1β, IFN-γ and IL-6, where the ethyl acetate fraction exhibited the highest activity. Additionally, untargeted and targeted metabolic profiling were performed using HPTLC-image analysis accompanied by multivariate analysis. This method has been implemented for the first time for the profiling of bioactive secondary metabolites in H. sabdariffa leaves correlated to its anti-inflammatory activity. Targeted metabolic profiling of the samples revealed the presence of lupeol, stigmasterol, β-sitosterol, ursolic acid, kaempferol, quercetin, stigmasterol glucoside, β-sitosterol glucoside, chlorogenic acid, rutin and kaempferol-3-O-glucoside, in addition to the tentative identification of a steryl glycoside and two flavonoids. Semi-quantitation of the identified compounds was established based on the peak areas of their corresponding spots on the TLC plates. Orthogonal projection to latent structures (OPLS) model correlated the HPTLC chromatographic data of the identified compounds with the anti-inflammatory activity revealing that kaempferol, quercetin, kaempferol-3-O-glucoside, flavonoid 1, flavonoid 2, rutin and chlorogenic acid are the chemical markers most contributing to the model. This is in line with their presence in high amount in the ethyl acetate fraction that displayed the greatest anti-inflammatory activity across all tested mediators. These results prove the potential of H. sabdariffa leaves as anti-inflammatory agent and the correlation of its content of flavonoids and phenolic acids to such activity.