Twitching motility is a mode of solid surface translocation that occurs under humid conditions on semisolid or solid surfaces, is dependent on the presence of retractile type IV pili and is independent of the presence of a flagellum. Surface translocation via twitching motility is powered by the extension and retraction of type IV pili and can manifest as a complex multicellular collective behavior that mediates the active expansion of colonies cultured on the surface of solidified nutrient media, and of interstitial colonies that are cultured at the interface between solidified nutrient media and an abiotic material such as the base of a petri dish or a glass coverslip. Here we describe two methods for assaying twitching motility mediated interstitial colony expansion in P. aeruginosa. The first method, the "Macroscopic Twitching Assay," can be used to determine if a strain is capable of twitching motility mediated interstitial colony expansion and can also be used to quantitatively assess the influence of mutation or environmental signals on this process. The second method, the "Microscopic Twitching Assay," can be used for detailed interrogation of the movements of individual cells or small groups of bacteria during twitching motility mediated colony expansion.