Cell assay that evaluates a biologics’ potency and efficacy is an important part in the discovery and development of drug candidates. However, it requires regular maintenance of cell cultures, and the cell assays can only be performed when the cells have reached 70-80% confluency. By instead using assay-ready cells, the drugs can be screened at any time by simply thawing the cells. This creates a more flexible assay, while saving time, labor and materials in addition to removing day-to-day variability. In this report, the freezing conditions for HEK-Blue™ CD40L cells are evaluated using the assay-ready cell method compared to a continuous culture. Via colorimetric detection, the CD40 receptors’ activation can be determined and a dose-response curve of a CD40 agonist can be produced. The optimal freezing condition for the assay-ready cells were determined to be 10% DMSO and a cell concentration between 1-30 million cells/mL. After reproducibility, robustness and screening tests, it could be concluded that the method generally produced results that had no significant difference to a continuous culture. Some of the assay-ready cells display a higher background which can affect the value of the efficacy. The source of the background will have to be evaluated in future studies. The potency, on the other hand, is stable regardless of cell method or high background.