Automated high-throughput methods that support tracking of mammalian cell growth are currently needed to advance cell line characterization and identification of desired genetic components required for cell engineering. Here, we describe a high-throughput noninvasive assay based on plate reader measurements. The assay relies on the change in absorbance of the pH indicator phenol red. We show that its basic and acidic absorbance profiles can be converted into a cell growth index consistent with cell count profiles, and that, by adopting a computational pipeline and calibration measurements, it is possible to identify a conversion that enables prediction of cell numbers from plate measurements alone. The assay is suitable for growth characterization of both suspension and adherent cell lines when these are grown under different environmental conditions and treated with chemotherapeutic drugs. The method also supports characterization of stably engineered cell lines and identification of desired promoters based on fluorescence output.