In preclinical drug development, the success rate of carrying candidates from phase I trials to final approval is as low as ~10%. Initially, compounds hitting specific molecular targets are selected via testing on permanent cell lines, xenotransplants, and animal experimentation. However, numerous failed clinical trials demonstrate that these models lack sufficient predictive power. In contrast to established cell lines altered by extended periods in tissue culture, freshly isolated cancer cells may represent original characteristics. For typical solid tumors, access to representative tumor cells is difficult, although pleura-derived tumor cells of advanced lung cancer patients may provide a notable exception to obtain original cell suspensions. These lung cancer cells can be used directly or upon the establishment of cell lines, which can be obtained rapidly at a high take rate due to the aggressive nature of these tumors. In contrast to established permanent cell lines, freshly isolated cells exhibit higher resistance and may more accurately predict responses. Furthermore, these cell lines can be validated within the framework of functional precision medicine for personalized chemotherapy by comparing the in vitro chemosensitivity profile of cells to clinical responses for specific inhibitors directed at mutated drivers such as Kirsten rat sarcoma viral oncogene homolog (KRAS), epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), c-ros oncogene 1, receptor tyrosine kinase (ROS1), and neurotrophic tropomyosin receptor kinase (NTRK) oncogenes in non-small cell lung cancer (NSCLC). This review discusses the use of freshly cultivated lung cancer cells from pleural effusions and highlights their value in generating clinically relevant predictive data, in contrast to permanent cell lines.