The advent of interferon-gamma release assays (IGRAs) provides new options for detection of latent tuberculosis infection (LTBI). This is particularly relevant to healthcare workers (HCWs), who are at higher risk of infection, but who have often also been vaccinated. In this article, we discuss the role of IGRAs for the diagnosis of LTBI in various healthcare settings. A search was performed for studies that reported data on IGRAs in HCWs in the last 18 years. Twenty-two studies met the inclusion criteria. IGRAs showed poor agreement with the tuberculin skin test (TST), except in countries with high incidences of tuberculosis (TB), but generally correlated better with markers of exposure to TB including during contact investigation. The T-SPOT.TB assay has not been adequately assessed in HCWs; the few studies available showed enhanced specificity of T-SPOT.TB when compared to TST. This review confirms the utility of IGRAs as important tools in the prevention and control of tuberculosis in healthcare settings.