Cancer is one of the leading causes of death in the world, where cervical cancer is the fourth leading cause of death in women. Existing treatments, although effective, have various adverse effects and often require a combination of two or more therapies. Previous studies have shown that different plant species of the genus Tournefortia sp. have traditionally been used against various diseases, including cancer, but it has not been possible to identify the specific compounds potentially responsible for this activity.Performing a bio-guided fractionation of Tournefortia hirsutissima L. extract, using Fast Centrifugal Partition Chromatography (FCPC) for the separation and identification of compounds with cytotoxic effects on cervix cancer cells (HeLa) and non-tumoral keratinocytes cells (HaCat).A bioactivity-guided assay was conducted to evaluate the cytotoxic activity against cervical cancer cells (HeLa) and non-tumoral cells (HaCat) of THL fractions obtained by Fast Centrifugal Partition Chromatography (FCPC).Pools 3 and 4, rich in 6,10,14-trimethylpentadecan-2-one and γ-Sitosterol, respectively, showed the strongest cytotoxic activity on cervical tumor cells HeLa with minimal effects on non-tumoral (HaCat) cell viability. Moreover, the formation of apoptotic bodies was observed in HeLa cells treated with 6,10,14-trimethylpentadecan-2-one, suggesting a possible programmed cell death mechanism.It was demonstrated that FCPC was a suitable technique to separate and identify the potential compounds responsible for the cytotoxic properties of THL extract. Likewise, 6,10,14-trimethylpentadecan-2-one, and γ-sitosterol can be considered potential therapeutic molecules against cervical cancer.Copyright © 2024 Elsevier B.V. All rights reserved.