The RAFT technique has been increasingly used to generate polymers for potential biological applications. However, to-date, the toxicity of the RAFT-polymers has received limited attention. In this study, the in vitro cytotoxicity of three different, RAFT-synthesized, water-soluble polymers was investigated using three different adherent cell lines via CellTiter-Blue cell viability and the cytosolic enzyme lactate dehydrogenase (LDH) cytotoxicity assays. In brief, P(OEG-A) and P(OEG-MA) samples bearing ?-dithiobenzoate or ?-trithiocarbonate end groups and varying P(HPMA) samples bearing ?-dithiobenzoate, ?-trithiocarbonate, or non-RAFT end groups, were investigated using Chinese hamster ovary cells (CHO-K1), mouse macrophage cells (Raw264.7), and mouse fibroblast cells (NIH3T3). Any changes in the morphology of the cells after treatment with polymers were monitored via microscopy. The cytotoxicity of the polymers after treatment with metabolic liver enzymes was also evaluated. The average viability of CHO-K1 and NIH3T3 cells treated with dithiobenzoate- and trithiocarbonate-ended OEGbased polymers (1000 ?M) for 24 h was close to 100 . The RAW264.7 cells were slightly more sensitive when incubated with dithiobenzoate-ended polymers (cell viability above 73 ) for 24 h. The viability of the cells after 3 days of incubation with the polymers either slightly decreased or showed no change with respect to the viabilities obtained after 1 day of incubation. Analyses of cell morphology and cell membrane integrity via microscopy and a LDH assay confirmed the cell viability results obtained via CellTiter-Blue Assay. Unexpectedly, dithiobenzoateended P(HPMA) (at 1000 ?M) exhibited high cytotoxicity after 24 h with all three cells lines. Further investigation of various P(HPMA) samples revealed that trithiocarbonate-ended and HPMA-capped P(HPMA)s at the same concentration were nontoxic over the same period of time. Also, dithiobenzoate-ended P(HPMA) at low concentrations (e200 ?M) can be tolerated by the cells tested.