We developed a cell-based assay based on the spin-assisted layer-by-layer (LbL) assembled polyelectrolyte matrix platforms. Three types of human breast epithelial cell lines including normal cells (184B5), noncancerous fibrocystic disease cells (MCF 10F), and metastatic cancerous cells (CAMA-1) were cultured, analyzed, and compared in parallel on various LbL-assembled polymer films. Poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) electrolyte polymers were used as the basic building units to form various LbL polyelectrolyte matrices. The mechanical rigidity, surface charge, and biorecognition property of the LbL platforms were controlled by tailoring the LbL surface, thermal cross-linking, and protein modification. Cellular phenotypic changes in adhesion, proliferation, and morphology on these LbL films were characterized and analyzed for the three different cell types. Our analysis results indicate that the cellular phenotype can be controlled by taking advantage of different surface charge, mechanical property, and biological modification (i.e., fibronectin in this case) of the LbL multilayer platforms. Importantly, cell phenotypical quantification results show that the cell spreading area per cell and optical density are useful parameters in distinguishing metastatic cancer cells from normal or fibrocystic disease cells on these LbL films. These LbL-based cell assay platforms have a potential for the development of various disease diagnostic cell assays.