Nanomedicines are enjoying a widespread popularity realizing their intriguing potential to solve drug delivery obstacles. Assessment of major quality attributes of nanocarriers is a crucial process for approving their therapeutic outcomes. Disparate assessment methods that recently encompassed cell line technique were employed . Routinely, a cell line model was viewed as an excellent platform for gene and vaccine deliveries. However, its application in pharmaceutical assessment of nanocarriers was not so far overviewed. This review provides a meticulous look at cell culture implementations in evaluation of major quality attributes of nanocarriers, including oral permeability, cytotoxicity and efficiency of tumor targeting. Among others, cell culture technique strikes the right balance between predictability and throughput. It could circumvent drawbacks of in-vivo and in-vitro techniques while gathering privileges of both. Imperative pharmaceutical considerations demanded for proper application of this technique were emphasized. Furthermore, challenges encountered in assessment of versatile nanocarriers were highlighted with proposed solutions. Finally, future research perspectives in this theme issue were suggested.