Background: Oncogenic structural rearrangements (SR) in ALK, RET and ROS1 are well-described in lung cancer, and confer sensitivity to targeted therapy. SR disrupting the 3’UTR of PD-L1 gene have been reported in multiple cancer types and can potentially predict response to checkpoint immunotherapy. An amplicon-based next-generation sequencing (NGS) platform technology (AmpliMARK), previously optimized for detection of single nucleotide variations (SNVs), microsatellite instability and viral DNA, was extended to the multiplex detection of SR in ALK, RET, ROS1 and PD-L1 in cell-free DNA (cfDNA) and tumor tissue DNA. Methods: A hybrid primer-extension and adapter-ligation based method allowing detection of SR in a fusion-partner agnostic manner was utilized for multiplex target capture of genomic regions of ALK, RET, ROS1 and PD-L1 SR. Analytical validation was performed using admixtures of fragmented genomic DNA from an ALK SR-positive cell line, commercial standards containing RET and ROS1 SR, and synthetic PD-L1 SR gene constructs. Clinical performance was assessed in cfDNA samples from lung cancer patients and tumor tissue DNA samples from natural killer(NK)/T-cell lymphoma patients. Results: Detection of SR could be achieved to an allele frequency detection limit of 0.5% with sensitivity of 89.5% and specificity of 100% in admixture samples mimicking cfDNA. In an unselected series of 374 lung cancer cases, actionable SR for ALK, RET and ROS1 were detected in cfDNA of 9 samples, for an overall detection rate of 2.4%, and 1.8% (3 out of 168) when restricted to treatment-naive lung cancer cases only. In 29 NK/T-cell lymphoma tumor tissue samples, 9 samples were positive for PD-L1 SR, which were orthogonally confirmed by whole-genome sequencing, targeted sequencing or Sanger sequencing for a concordance rate of 100% across all samples. For 1 NK/T-cell lymphoma tumor tissue sample where matched plasma was available, the same PD-L1 SR was also detected in cfDNA. Conclusions: We have demonstrated and validated a comprehensive amplicon-based NGS assay for ultrasensitive multiplex detection of structural rearrangements in ALK, RET, ROS1 and PD-L1 across both cfDNA and tumor tissue DNA in analytical and clinical contexts. Ongoing studies will further evaluate the performance and utility of this assay across a larger number of clinical samples for the detection of these SR as well as additional cancer-associated SR involving NTRK1/2/3, FGFR2/3 and TMPRSS2.