A real-time polymerase chain reaction (PCR) assay targeting the internal transcribed spacer 2 region of the ribosomal RNA gene was developed for the detection of Isospora belli DNA in fecal samples, including an internal control to detect inhibition during the amplification process. The assay was performed on species-specific DNA controls (n = 27) and a range of positive (n = 21) and negative (n = 120) stool samples, and achieved 100% specificity and sensitivity. The simple fecal sample collection procedure, the high-throughput potential, and the possibility of quantification makes the I. belli real-time PCR assay a powerful diagnostic tool for epidemiologic studies with possibilities for extension to other helminthes and protozoa using additional molecular targets. In addition, this Isospora PCR could augment the clinical laboratory diagnosis of isosporiasis, in particular, in patients with a travel history to developing countries.