Organ-on-a-chip devices combine microfabrication, tissue engineering, and microfluidics to recreate physiologically relevant microenvironments for in vitro studies. In this work, we validated a dynamic 2D breast-on-a-chip microfluidic bioassay operated at a controlled infusion rate of 20 µL/h to assess anticancer drug responses under defined flow conditions. Using Palbociclib as a reference compound, we evaluated proliferation, viability/apoptosis, cytoskeleton organization, and differential processing of the resistance-associated marker PARP1 in MCF-7 and T47D breast cancer cells. Under dynamic microfluidic conditions, Palbociclib induced dose-dependent effects, with the higher concentration (20 µM) consistently reducing cell proliferation and viability and increasing late apoptosis compared to 10 µM Palbociclib. Cytoskeletal disorganization was observed at both concentrations, while differential PARP1 processing patterns between MCF-7 and T47D cells were detected across doses. These responses are consistent with known effects of CDK4/6 inhibition and were reproducibly captured under controlled flow conditions. Overall, our results demonstrate that this breast-on-a-chip microfluidic model provides a reproducible and physiologically relevant in vitro platform for integrated assessment of drug efficacy and resistance-associated markers under dynamic perfusion.