Direct visualization chambers are considered the gold standard for measuring and analyzing chemotactic responses, because they allow detailed analysis of cellular behavior during the process of chemotaxis. We have previously described the Insall chamber, an improved chamber for measuring cancer cell chemotaxis. Here, we describe in detail how this system can be used to perform two key assays for both fast- and slow-moving mammalian and nonmammalian cell types. This allows for the detailed analysis of chemotactic responses in linear gradients at the levels of both overall cell behavior and subcellular dynamics.