Construction and supply of cell-based reagents for in vitro plate-based screens are often highlighted as a bottleneck within drug discovery. Recent years have seen the successful application of both cryopreservation and automation to increase the capacity and flexibility of cell provision. However, routine cell culture remains a fixed experimental process that requires cells to be prepared and used at specific times. We have investigated the potential of reduced temperature incubation to be used as a simple methodology for stopping and starting cell growth and introduce further flexibility into cell provision. Our results show that incubation of CHOK1, HEK293, and 1321N1 cells at 23 degrees C arrested growth while maintaining cell viability. Recovery of these paused cells at 37 degrees C resulted in resumption of normal cell growth and target protein function. Experiments demonstrated that paused cells, expressing either a recombinant G-protein-coupled receptor or an ion channel, performed comparably with the equivalent continuously cultured cells in a 384-well cell-based assay. This simple technique offers the potential to introduce flexibility into cell culture experiments and processes that were previously considered to be fixed.