A series of novel vindoline-piperazine conjugates were synthesized by coupling 6 -substituted piperazine pharmacophores at positions 10 and 17 of alkaloid monomer vindoline through different types of linkers. The in vitro antiproliferative activity of the 17 new conjugates was investigated on 60 human tumor cell lines (NCI60). Nine compounds presented significant antiproliferative effects. The most potent derivatives showed low micromolar growth inhibition () values against most of the cell lines. Among them, conjugates containing [4-(trifluoromethyl)benzyl]piperazine () and 1-bis(4-fluorophenyl)methyl piperazine () in position 17 of vindoline were outstanding. The first one was the most effective on the breast cancer MDA-MB-468 cell line ( = 1.00 μM), while the second one was the most effective on the non-small cell lung cancer cell line HOP-92 ( = 1.35 μM). The CellTiter-Glo Luminescent Cell Viability Assay was performed with conjugates , , and on non-tumor Chinese hamster ovary (CHO) cells to determine the selectivity of the conjugates for cancer cells. These compounds exhibited promising selectivity with estimated half-maximal inhibitory concentration () values of 2.54 μM, 10.8 μM, and 6.64 μM, respectively. The obtained results may have an impact on the design of novel vindoline-based anticancer compounds.