Alternative DNA structures, such as Z-DNA, G-quadruplexes, and mirror repeats, have shown potential involvement in cancer etiology. NBBC (Non-B DNA Burden in Cancer) is a web-based tool designed for quantifying and analyzing non-B DNA motifs within a cancer context. Herein, we provide a step-by-step protocol for employing NBBC, starting with data input and proceeding through the quantification and normalization of non-B DNA motifs that result in calculation of non-B burden. With detailed instructions for performing various motif-centric analyses based on cancer gene signatures, including DNA damage repair and response pathways for genomic stability, and other sample-level gene mutation signatures, users can explore non-B associative correlations within current cancer biology. We provide additional details on input queries into NBBC, interpret the quantitative results, and apply normalization techniques to ensure accurate comparisons across different genomic regions and non-B DNA structures. NBBC offers a powerful and user-friendly interface for the cancer research community. This chapter serves as an essential, enhanced instructional guide for researchers to leverage NBBC in their cancer biomarker investigations for an understanding of the potential role of non-B DNA in contributing to them.