A real-time PCR method targeting the small subunit of the rRNA gene was developed for the detection of Strongyloides stercoralis DNA in faecal samples, including an internal control to detect inhibition of the amplification process. The assay was performed on a range of well-defined control samples (n = 145), known positive faecal samples (n = 38) and faecal samples from a region in northern Ghana where S. stercoralis infections are highly endemic (n = 212), and achieved 100% specificity and high sensitivity. The use of this assay could facilitate monitoring the prevalence and intensity of S. stercoralis infections during helminth intervention programs. Moreover, the use of this assay in diagnostic laboratories could make the introduction of molecular diagnostics feasible in the routine diagnosis of S. stercoralis infections, with a two-fold increase in the detection rate as compared with the commonly used Baermann sedimentation method. © 2008 Royal Society of Tropical Medicine and Hygiene.