Neutrophils are the initial responders to bacterial infection or other inflammatory stimuli and comprise a key component of the innate immune response. In addition to their unique morphology and antimicrobial activity, neutrophils are characterized by the ability to migrate rapidly up shallow gradients of attractants in vivo. The directed migration of neutrophils, referred to as chemotaxis, requires the temporal and spatial regulation of intracellular signaling pathways allowing the neutrophil to detect a gradient of attractant, polarize, and migrate rapidly toward the highest concentration of the chemoattractant. A challenge to understanding neutrophil chemotaxis is the inherent difficulty encountered when working with primary neutrophils, which are difficult to purify in the resting state, are not easily transfected, are terminally differentiated, and have a short life span after purification. Here we discuss neutrophil purification methods and chemotaxis assays and provide methodology for working with a neutrophil-like cell line, the HL-60 promyelocytic leukemia cell line. We also discuss methods for HL-60 transfection using retroviral approaches and chemotaxis assays used with differentiated HL-60 cells.