Reliable diagnostic markers for Bladder Pain Syndrome/Interstitial Cystitis (IC) currently are not available. This study evaluated the feasibility of diagnosing IC in humans and domestic cats from the spectra of dried serum films (DSFs) using infrared microspectroscopy. Spectra were obtained from films from 29 humans and 34 domestic cats to create classification models using Soft Independent Modeling by Class Analogy (SIMCA). Ultrafiltration of serum improved discrimination capability. The classification models for both species successfully classified spectra based on condition (healthy/sick), and a different set of masked spectra correctly predicted the condition of 100% of the subjects. Classification required information from the 1500-1800 cm -1 spectral region to discriminate between subjects with IC, other disorders, and healthy subjects. Analysis of cat samples using liquid chromatography-mass spectroscopy revealed differences in the concentration of tryptophan and its metabolites between healthy and affected cats. These results demonstrate the potential utility of infrared microspectroscopy to diagnose IC in both humans and cats. © 2009 Royal Society of Chemistry.