Non-canonical DNA structures add a dynamic layer of genome regulation beyond the classical double helix. I-motifs, cytosine-rich four-stranded DNA structures, are increasingly recognized as context-dependent regulators of genome function. Recent methodological advances, including i-motif-specific antibodies, in-cell NMR, and genome-wide profiling, have enabled their detection and functional interrogation. Here, we review progress in understanding i-motif formation, stability, and protein interactions, highlighting parallels and contrasts with G-quadruplex structures. We also discuss technical limitations, strategies for improving structure-specific resolution, and future opportunities to integrate biochemical, genomic, and imaging approaches to clarify the biological relevance and therapeutic potential of i-motifs.