As previously reported, a series of Betulinic acid (1) derivatives containing 2,4- dinitrophenylhydrazone (2,4-DNPH) moiety was synthesized and evaluated for their anticancer potential. Aldehyde 2 was the most cytotoxic compound (IC50 1.76-2.51 µM), while 3 was the most selective compound (5-7 times) towards cancer cells. 2,4-DNPH derivatives were able to arrest G0/G1 phase of the cell cycle of A549 cell line. In this report, Caspases-3, -8 and -9 activation was assayed in order to contribute to the elucidation of the mechanism of action of these Betulinic acid 2,4-DNPH derivatives. A549 lung cancer cells were treated with IC80 concentrations of compounds 1, 2 and 3 during 24, 48 and 72 h. Cells (1.0x106 cell/mL) were incubated with caspase-3, -8 and -9 inhibitors and analyzed by flow cytometry. Compound 1 activated around 90% caspases-3 and -8 after 24 h treatment. After 48 h, all caspases were practically 100% activated. Compounds 2 and 3 did not activate significantly any caspases after 24 h. Compound 2 was able to activate significantly caspases (ca. 50 %) only after 72 h. 3 substantially activated caspases-3 and -8 after 48 h, but after 72 h activation was up to 75%. 2,4- DNPH moiety plays an important role for the cytotoxicity of these molecules and also for the activation of caspases and apoptosis induction. By another hand, the introduction of 2,4-DNPH moiety in 1 altered significantly the kinetics of the molecules, since the mechanism of action of the derivatives was slower than the precursor.