Drug induced accumulation of phospholipid (phospholipidosis) is associated with clinical toxicities and animal testing is still required for a predictive risk assessment. However, in silico models and cell-based assays can help to evaluate the phospholipidosis potential of a drug at an early stage of preclinical development and may reduce animal testing. Cells used in cell based assays are usually prepared fresh from a maintenance culture. However, when handled carefully and cryopreserved according to optimized protocols, the functionality of the cells can be maintained and is fully restored after thawing of the cells. Assay ready cells which are frozen at highly viable and functional state are ready to use directly after thawing and don’t need to be cultivated or passaged before. We used assay ready HepG2 cells in phospholipidosis assay to demonstrate that assay ready cells are equal to fresh cells from a growing culture. This approach provides a higher flexibility, greater reliability and increase in assay reproducibility.