Biologically active compounds of natural origin, such as betulin, are a source of obtaining new medicinal substances. The presence of chemically active hydroxyl groups in the betulin structure at C-3 and C-28 positions enables esterification with dicarboxylic acid anhydrides or carboxylic acids. As a result of a four-step synthesis, difunctional betulin derivatives were obtained, which were evaluated for their antiproliferative activity against the following human cell lines: leukemia (MV4-11), (A549), breast cancer (MCF-7), prostate adenocarcinoma (PC-3), colon cancer (HCT116), pancreatic cancer (MiaPaca-2), and melanoma (Hs294T). The target 3-carboxyacyl-28-alkynyloyl betulin derivatives showed significant antiproliferative activity against MV4-11 cells. For 3-carboxyacylbetulins and their selected alkynyl derivatives, studies to investigate the effect on the cell cycle and apoptosis process, as well as drug similarity analysis, were performed.