Formulation optimization from the early steps plays an important role in the success of drug delivery system development. To optimize nano-sized micelles based on poly(ethylene glycol)-b-poly(lactide)-b-poly(ethylene glycol) (PEG-PLA-PEG) triblock copolymers, the effect of PLA composition on the physicochemical properties of micelles was studied. Doxorubicin (DOX) was used to be encapsulated into micelles. In vitro studies on the safety of triblock copolymers and cytotoxicity of DOX-loaded micelles compared to free DOX were done, using MDA-MB-231 cells. Critical micelle concentration and micelle size were found to be linearly dependent on the PLA molecular weight (MW). In addition, it was shown that long PLA-containing micelles had low stability. The sizes of DOX-loaded micelles were bigger than those of empty micelles. The loading amount of DOX into micelles as well as the release rate of DOX from micelles depended on the PLA MW. Triblock copolymers themselves did not show any toxicity over a wide range of concentration. DOX-loaded micelles killed more tumor cells than free DOX. In summary, difference in hydrophobicity can be a critical factor to determine the physicochemical properties of micelles composed of PEG-PLA-PEG, and thus, it can affect the drug delivery efficacy of micelles. After considering all related factors, PEG-PLA-PEG 2 kDa-6 kDa-2 kDa seemed to be the best polymer for further studies.