The prevalence and severity of skin cancer, specifically malignant melanoma, among Caucasians remains a significant concern. Natural compounds from plants have long been explored as potential anticancer agents. Betulinic acid (BI) has shown promise in its therapeutic properties, including its anticancer effects. However, its limited bioavailability has hindered its medicinal applications. To address this issue, two recently synthesized semisynthetic derivatives, N-(2,3-indolo-betulinoyl)diglycylglycine (BA1) and N-(2,3-indolo-betulinoyl)glycylglycine (BA2), were compared with previously reported compounds N-(2,3-indolo-betulinoyl)glycine (BA3), 2,3-indolo-betulinic acid (BA4), and BI. These compounds were evaluated for their effects on murine melanoma cells (B164A5) using various in vitro assays. The introduction of an indole framework at the C2 position of BI resulted in an increased cytotoxicity. Furthermore, the cytotoxicity of compound BA4 was enhanced by conjugating its carboxylic group with an amino acid residue. BA2 and BA3, with glycine and glycylglycine residues at C28, exhibited approximately 2.20-fold higher inhibitory activity compared to BA4. The safety assessment of the compounds on human keratinocytes (HaCaT) has revealed that concentrations up to 10 µM slightly reduced cell viability, while concentrations of 75 µM resulted in lower cell viability rates. LDH leakage assays confirmed cell membrane damage in B164A5 cells when exposed to the tested compounds. BA2 and BA3 exhibited the highest LDH release, indicating their strong cytotoxicity. The NR assay revealed dose-dependent lysosome disruption for BI and 2,3-indolo-betulinic acid derivatives, with BA1, BA2, and BA3 showing the most cytotoxic effects. Scratch assays demonstrated concentration-dependent inhibition of cell migration, with BA2 and BA3 being the most effective. Hoechst 3342 staining revealed that BA2 induced apoptosis, while BA3 induced necrosis at lower concentrations, confirming their anti-melanoma properties. In conclusion, the semisynthetic derivatives of BI, particularly BA2 and BA3, show promise as potential candidates for further research in developing effective anti-cancer therapies.