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The transposase-accessible chromatin using sequencing (ATAC-seq) offers a simplified approach to detect chromatin changes in cancer cells after genetic intervention and drug treatment. Here, we present an optimized ATAC-seq protocol to elucidate chromatin accessibility changes at the epigenetic level in head and neck squamous cell carcinoma cells. We describe steps for cell lysate preparation, transposition, and tagmentation, followed by library amplification and purification. We then detail next-generation sequencing and data analysis. For complete details on the use and execution of this protocol, please refer to Buenrostro et al., Chen et al.,.