Background: Blockade of PD-1/PD-L1 immune checkpoint pathway emerged as promising novel therapeutic strategy for cancer. Predictive biomarkers for response to anti-PD1 therapy are lacking. Because therapy with checkpoint inhibitors is cost intensive, noninvasive tools for early prediction of responders are of major interest. We assessed gene expression of PDL1 in CTCs at baseline and posttreatment in HNSCC patients (pts). Methods: 70 pts with locally advanced (n = 58) or recurrent/metastatic (n = 12) HNSCC were included in this analysis. Patients with locally advanced disease were treated with cisplatin chemoradiotherapy +/- TPF induction chemotherapy (IC). We assessed PDL1 expression at baseline, after completion of induction chemotherapy, at end of chemoradiotherapy and at relapse. We quantified PDL1 gene transcripts in immunomagnetically positively selected CTCs and 20 healthy individuals. A quantitative real time RT-qPCR assay for PDL1 was developed based on de novo in-silico design of primers and Taqman probes. The specificity was first tested by homology searches in the nucleotide database (NCBI, nucleotide BLAST). To assess univariate differences of study parameters according to PDL1 expression chi-square test was used for the categorical clinicopathological variables, while patients’ survival curves according to PDL1expression were generated by Kaplan-Meier analysis and tested for significance using the Mantel-Cox log-rank test. Results: From 88 total evaluable CTC samples, 26 of 46 were PDL1+ at baseline, 6 of 17 post-IC, 11 of 23 at end of treatment and 1 of 2 at relapse. The assay sensitivity was evaluated using external quantification calibrators ranging from 105 copies/μL to 10 copies/μL. There was trend (p = 0.07) for adverse association between PDL1 expression at baseline and progression-free survival. Conclusions: We report for the first time a highly sensitive, specific and reproducible quantitative real-time RT-qPCR assay for the assessment of PDL1 expression in CTCs. The assay is currently validated in CTCs isolated from a large number of HNSCC pts. Serial PDL1 expression assessment has potential to select and monitor pts for PD-1 targeted therapies.