Cell monolayers underpin the discovery and screening of new drugs and allow for fundamental studies of cell biology and disease. However, current cryopreservation technologies do not allow cells to be stored frozen while attached to tissue culture plastic. Hence, cells must be thawed from suspension, cultured for several days or weeks, and finally transferred into multiwell plates for the desired application. This inefficient process consumes significant time handling cells, rather than conducting biomedical research or other value-adding activities. Here, we demonstrate that a synthetic macromolecular cryoprotectant enables the routine, reproducible, and robust cryopreservation of biomedically important cell monolayers, within industry-standard tissue culture multiwell plates. The cells are simply thawed with media and placed in an incubator ready to use within 24 h. Post-thaw cell recovery values were >80% across three cell lines with low well-to-well variance. The cryopreserved cells retained healthy morphology, membrane integrity, proliferative capacity, and metabolic activity; showed marginal increases in apoptotic cells; and responded well to a toxicological challenge using doxorubicin. These discoveries confirm that the cells are "assay-ready" 24 h after thaw. Overall, we show that macromolecular cryoprotectants can address a long-standing cryobiological challenge and offers the potential to transform routine cell culture for biomedical discovery.