A potent nontoxic antitumor drug, 2-hydroxyoleic acid (6, 2OHOA) used for membrane lipid therapy, was selected as a self-assembly inducer due to its ability to form nanoparticles (NPs) in water. For this purpose, it was conjugated with a series of anticancer drugs through a disulfide-containing linker to enhance cell penetration and to secure drug release inside the cell. The antiproliferative evaluation of the synthesized NP formulations against three human tumor cell lines (biphasic mesothelioma MSTO-211H, colorectal adenocarcinoma HT-29, and glioblastoma LN-229) showed that nanoassemblies16–22a,bNPsexhibit antiproliferative activity at micromolar and submicromolar concentrations. Furthermore, the ability of the disulfide-containing linker to promote cellular effects was confirmed for most nanoformulations. Finally,17bNPinduced intracellular ROS increase in glioblastoma LN-229 cells similarly to free drug8, and such elevated production was decreased by pretreatment with the antioxidantN-acetylcysteine. Also, nanoformulations18bNPand21bNPconfirmed the mechanism of action of the free drugs.