Spirochaetes are spiral-shaped, slender bacteria that can be free-living or associated with a host. There are six genera of spirochaetes found in various environments, including the human oral cavity, gastrointestinal tracts of humans and mammals, insects, and marine environments. Some spirochaetes, such as Treponema (which causes Syphilis), Borrelia (which causes Lyme disease and relapsing fever), Leptospira (which causes leptospirosis), and Brachyspira (which causes intestinal spirochaetosis), are serious pathogens for humans and can cause severe diseases. Despite the identification of several potential virulence factors in these bacteria, the mechanisms of pathogenesis remain unclear. Understanding the molecular and cellular pathogenesis of spirochaetes is still in its infancy. This Methods Collection aims to demonstrate standardized protocols and techniques to address the recent and advanced challenges of studying host-pathogen interaction-mediated pathogenesis.