Bisindole alkaloids, also known as dimeric indole alkaloids, are composed of two monomeric indole units linked by C–C, C–O–C, or C–N bonds. These compounds are predominantly found in plant families such as Loganiaceae, Apocynaceae, and Rubiaceae. Compared to their monomeric counterparts, bisindole alkaloids exhibit significantly enhanced biological activity. Catharanthus species, particularly C. roseus, are a rich source of pharmacologically active bisindole alkaloids, including vinblastine and vincristine—two widely used anticancer agents. The near-simultaneous discovery of vinblastine remains a landmark achievement in alkaloid chemistry. Traditionally, the isolation, fractionation, and purification of bisindole alkaloids involve time-consuming, labor-intensive, and costly procedures. However, advanced analytical techniques such as HPTLC, HPLC, capillary electrophoresis–mass spectrometry (CE–MS), and HPLC–MS/MS have greatly improved sensitivity, specificity, and efficiency in both qualitative and quantitative analyses. This chapter provides a comprehensive overview of various modern analytical approaches employed for the detection and characterization of bisindole alkaloids, highlighting the benefits of using cutting-edge chromatographic and mass spectrometric methods over traditional techniques.