The bacterial type VI secretion system (T6SS) is a secretory apparatus encoded by many Gram-negative bacteria. The T6SS facilitates the secretion and injection of toxic effector proteins into host cells, providing a competitive advantage to bacteria encoding this machinery. The activity of the T6SS can be monitored by probing for the conserved tubule component Hcp, which is secreted to the supernatants by the T6SS. Detection of Hcp in culture supernatants is indicative of an active T6SS, but this secretion system is often tightly regulated or inactive under laboratory conditions and different bacterial strains display differing Hcp secretion phenotypes. Herein, we describe an enzyme-linked immunosorbent assay (ELISA) and colony blot methods to facilitate large-scale screening of isolates for Hcp secretion and, thus, T6SS activity.