AbstractFormaldehyde (HCHO) is a simple and highly reactive human metabolite but its biochemistry is poorly defined. A limiting factor in HCHO research is lack of validated quantification methods for HCHO relevant to biological samples. We describe spectroscopic studies on a reported fluorescence-based HCHO detection method involving its reaction with ampicillin. The results validate the structure and fluorescence properties of the HCHO-ampicillin reaction product. However, the same adduct is observed after reaction of ampicillin with glyoxylate. Related fluorophores were formed with other biologically relevant carbonyl compounds. Overall, our studies suggest the ampicillin method is not reliable for selective detection and quantification of HCHO in biological samples.