Lithocholic acid (LA) as the main component of bile and its derivatives are biologically active compounds - they are proteasome regulators, activate the vitamin D receptor and enhance the interaction of cholecalciferols with the receptor, exhibit inhibitory activity against DNA polymerases β (pol β), and appear antibacterial and antitumor effects. We have previously shown that the synthesized hybrid molecules based on steroids and cis-unsaturated acids are apoptosis inducers in cell cultures Jurkat, K562, U937, HeLa, HEK293 and inhibit in vitro the relaxation of supercoiled DNA induced by topoisomerase I. In the continuation of these studies, we synthesized the conjugates LA and (5Z, 9Z)-tetradeca-5,9-dienedioic acid, which are linked through ethylene glycol and diaminoalkane spacers of different lengths, and also studied the antitumor activity of the synthesized compounds. The synthesis of the target hybrid molecules based on LA and (5Z,9Z)-tetradeca-5,9-dienedioic acid, was carried out in two approaches: via 3-О-acetation of LA using a dicarboxylic acid, and then modified the LA carboxyl group at the С-24 position. For all synthesized compounds, the in vitro antitumor activity was evaluated for the first time on Jurkat, K562, HEK293, HELA, and U937 cell lines, using the Guava Nexin Reagent, Guava Cell Cycle and Guava ViaCount (Millipore) reagent kits, including the determination of IC50, the study of viability cells and influence on the cell cycle using flow cytometry.