Purpose To determine feasibility and safety of biopsy and repeat biopsy for assessment of programmed cell death ligand-1 (PD-L1) status. Materials and Methods This retrospective analysis reviewed 101 patients who underwent transthoracic core needle biopsy for the KEYNOTE-001 (MK-3475) clinical trial of pembrolizumab, an antiprogrammed cell death-1 therapy for non-small cell lung cancer, from May 2012 to September 2014. Sixty-one male patients (mean age, 66.1 years; range 36-83 years) and 40 female patients (mean age, 66.8 years; age range, 36-90 years) were included. Data collected included population characteristics, treatment history, target location, size, and depth from pleura. Adequacy of the tissue sample for diagnostic testing and rates of biopsy-related complications were assessed. Statistical analysis was performed by using univariate and multivariate generalized linear models to determine significant risk factors for biopsy complications. Results A total of 110 intrathoracic biopsies were performed, and 101 (91.8%) were performed as repeat biopsies subsequent to a previous percutaneous or bronchoscopic biopsy or previous surgical biopsy or resection. More than 84.5% (93 of 110) of biopsies were performed in patients who had undergone previous local or systemic therapy. Specimens were adequate for evaluation of PD-L1 expression in 96.4% of biopsies. Procedure-related complications occurred in 28 biopsies (25.4%); pneumothorax was most common (22.7%). Overall mean number of core needle biopsy samples obtained was 7.9 samples. Conclusion Image-guided transthoracic core needle biopsy is an effective method for obtaining tissue for PD-L1 expression analysis. © RSNA, 2017.