The ability to test stool for laxatives is an important part of patient care in some clinical circumstances. Some patients take or are given laxatives surreptitiously. Additionally, failure to take prescribed laxatives may result in treatment failure in children with constipation or encopresis. Although laboratory methods have been available to identify many laxatives in the stool, tests are not available for detecting polyethylene glycol (PEG)-based laxatives. PEG-based laxatives are frequently used in the treatment of children with constipation. We developed a mass spectrometry (MS)-based analysis for detecting PEG in stool and verified the technique in an adult volunteer. We then piloted the assay on stools from children taking PEG for constipation versus children with diarrhea who were not taking PEG. Eleven subjects with diarrhea and 8 receiving PEG were enrolled. Nine of the children with diarrhea and 7 receiving PEG were evaluated by MS. All 3 subjects with PEG who had a stool osmolal gap determined had elevated gaps. Stools of all 7 subjects with PEG were positive for PEG by MS, whereas none of the 9 subjects with diarrhea had stool positive for PEG. This new MS methodology to test stool for PEG is described. It is likely to prove useful in the documentation of surreptitious PEG administration and in evaluation of PEG treatment failure.