Balkan endemic nephropathy (BEN) is a peculiar renal disease affecting thousands of farmers living in the suburban areas of the Balkan countries. Emerging evidence suggested that BEN is an environmental disease caused by chronic food poisoning with aristolochic acid (AA). We have developed a sensitive liquid chromatography-tandem mass spectrometry method to detect urinary RNA-AA adducts. Results revealed high levels of RNA-AA adducts in the urine samples collected from AA-treated rats. To the best of our knowledge, this study is the first to report on the detection of urinary RNA-AA adducts. Compared with previous studies that quantified DNA-AA adducts, this method is more sensitive and user-friendly.