Strongyloidiasis is a neglected disease that is prevalent mainly in tropical and subtropical regions. It is caused by intestinal nematodes of the genus Strongyloides. Due to the rise in worldwide travel, infections are increasingly encountered in non-endemic regions. Diagnosis is hampered by insensitive and laborious detection methods. A universal Strongyloides species real-time PCR was developed with an internal competitive control system. The 95% limit of detection as determined by probit analysis was one larva per PCR equivalent to 100 larvae per 200 mg stool. The assay proved to be 100% specific as assessed using a panel of parasites and bacteria and thus might be useful in the diagnostic setting as well as for Strongyloides research.