Bortezomib (BTZ) is a proteasome inhibitor used for multiple myeloma and mantle cell lymphoma treatment. BTZ's aqueous in solubility is the main hindrance in its successful development as a commercial formulation. The main objective of the present study is to develop and characterize folic acid-glycine-poly-L-lactic acid (FA-Gly-PLA) based nanoformulation (NPs) to improve solubility and efficacy of BTZ.BTZ loaded FA-Gly-PLA NPs were prepared and characterized for size, zeta potential, in vitro studies such as release, kinetics modeling, hemolytic toxicity, and cell line-based studies (Reactive Oxygen Species: ROS and cytotoxicity).BTZ loaded NPs (BTZ-loaded FA-Gly-PLA) and blank NPs (FA-Gly-PLA) size, zeta, and PDI were found to be 110 ± 8.1 nm, 13.7 ± 1.01 mV, 0.19 ± 0.03 and 198 ± 9.01 nm, 8.63 ± 0.21 mV, 0.21 ± 0.08 respectively. The percent encapsulation efficiency (% EE) and percent drug loading (% DL) of BTZ loaded FA-Gly-PLA NPs was calculated to be 78.3 ± 4.1 and 12.38 ± 2.1. The Scanning Electron Microscopy (SEM) showed that NPs were slightly biconcave in shape. The in vitro release of BTZ from FA-Gly-PLA NPs resulted in the sustained manner. The prepared NPs were less hemolytic than BTZ.BTZ loaded Gly-PLA NPs apoptotic index was found to be much higher than BTZ but lesser than BTZ loaded FA-Gly-PLA against breast cancer cell lines (MDA-MB-231). ROS intracellular assessment assay indicated that BTZ and BTZ loaded FA-Gly-PLA NPs exhibited higher ROS production. Conclusively, the BTZ loaded FA-Gly-PLA NPs were able to encapsulate more BTZ than BTZ loaded Gly-PLA NPs and were found to be more effective as per as in vitro anti-cancer effect is concerned.