Circulating tumor cells (CTCs) hold great potential to answer key questions of how non-small cell lung cancer (NSCLC) evolves and develops resistance upon anti-PD-1/PD-L1 treatment. Currently, their clinical utility in NSCLC is compromised by a low detection rate with the established, Food and Drug Administration (FDA)-approved, EpCAM-based CellSearch System. We tested an epitope-independent method (Parsortix system) and utilized it to assess PD-L1 expression of CTCs from NSCLC patients. We prospectively collected 127 samples, 97 of which were analyzed with the epitope-independent system in comparison to the CellSearch system. CTCs were determined by immunocytochemistry as intact, nucleated, CD45 , pankeratins (K) cells. PD-L1 status of CTCs was evaluated from 89 samples. With the epitope-independent system, ≥1 CTC per blood sample was detected in 59 samples (61%) compared to 31 samples (32%) with the EpCAM-based system. Upon PD-L1 staining, 47% of patients harbored only PD-L1 CTCs, 47% had PD-L1 and PD-L1 CTCs, and only 7% displayed exclusively PD-L1 CTCs. The percentage of PD-L1 CTCs did not correlate with the percentage of PD-L1 in biopsies determined by immunohistochemistry ( = 0.179). Upon disease progression, all patients showed an increase in PD-L1 CTCs, while no change or a decrease in PD-L1 CTCs was observed in responding patients ( = 11; = 0.001). Our data show a considerable heterogeneity in the PD-L1 status of CTCs from NSCLC patients. An increase of PD-L1 CTCs holds potential to predict resistance to PD-1/PD-L1 inhibitors.