Ralstonia solanacearum, a gram-negative bacterium, is known to cause devastating wilting disease in many plant species. The bacterium’s pathogenesis relies on various virulence determinants, including twitching motility, facilitated by type IV pili appendages. Twitching motility is a density-dependent mechanism enabling bacterial movement on solid surfaces, crucial for biofilm formation and virulence. Understanding this motility is of utmost importance in studying the pathogen’s behavior. In this chapter, we present a simple and reproducible method to assess twitching motility in R. solanacearum. The method involves microliter spotting of a diluted bacterial suspension onto a solid agar growth medium, followed by microscopic observation of the early-stage microcolonies. Twitching motility becomes evident as finger-like projections, which merge and progressively spread in all directions, forming multilayered structures. The examination also reveals cell heterogeneity within colonies and the influence of bacterial concentration on microcolony shape, potentially attributed to quorum sensing. Our simplified protocol for assessing twitching motility in microcolonies using microliter spotting offers significant advantages over existing methods described in the literature, making it an effective tool for investigating twitching motility in bacterial pathogenesis. A detailed step-by-step description of the protocol is provided in this chapter, facilitating its adoption and application in future research.