The main in vitro methods for immunologists to analyse cells of the immune system are flow cytometry, PCR and various forms of immunoassay, such as ELISPOT. Together, these techniques enable measurement of different cell populations at the molecular and functional level (immuno-phenotyping), as well as quantification of immune responses, for example cytokine release. Although extremely powerful, these methodologies provide little insight into cell morphology or spatial interactions. Moreover, as ‘end-point’ measurements, they are not well suited to reporting on changes in biology over time. In this article, we examine real-time live-cell analysis as an enabling technology for immunology research, including case study data for a range of functional bioassays.