The growing interest in herbal and alternative medicines demands information on the toxicity risk assessment of the various plant extracts used in traditional medicines. The presence of a potential toxic phytochemicals in Tithonia diversifolia extracts necessitates toxicological evaluation of its biologically active constituents. The study evaluated the in vivo sub-chronic toxicity of the moderately polar fractions of T. diversifolia in a rat model. The ethyl acetate soluble portion from the methanol extract was separated by vacuum liquid chromatographic method. Three-dose levels- an observed adverse effect level (OAEL) of 2000 mg/kg, a no-observed adverse effect level (NOAEL) of 80 mg/kg and an intermediate dose of 500 mg per kg body weight of rats per day- were selected for a 28-day repeated dosing for the sub-chronic toxicological evaluation. The LC-MS dereplication of the active fractions showed the presence of sesquiterpene lactones such as diversifolin, diversifolin methylether, tagitinin A, tagitinin C-F, woodhousin, and orizatin and many unidentified peaks. There was a significant reduction (p < 0.05) in the weights of and food consumption by the rats dosed with OAEL of the fraction on week 1 which normalized during the subsequent weeks of the study. The histopathological examination showed a mild necrosis and degeneration of hepatocytes in the centrilobular areas of the rats treated with OAEL of the active VLC fraction. There was no T. diversifolia-related adverse toxicological events in rats with a 2000 mg/kg/day when dosed orally for 28 days.