Objectives: The urinary proteome is a potential easily accessible source of biomarkers for inflammatory bladder diseases, including interstitial cystitis. In the present study, we subjected rat urine to multiplex cytokine analysis in an attempt to identify an inflammatory signature of the temporal course of cyclophosphamide (CYP)-induced cystitis. Methods: Rat urine was collected for 12 hours after CYP injection (150 mg/kg) for multiplex analysis of 14 cytokines by a multiple antigen bead assay (Luminex 100 IS). Urine from each void was collected, and the voiding frequency was determined. The bladder tissue was analyzed for cytokines levels and histologic evidence of inflammation. Results: Significant changes were noted in the urine levels of all cytokines with respect to baseline at 2, 4, 6, and 10 hours after CYP injection. Elevation was noted at all times for most cytokines, except for monocyte chemotactic protein-1, which had a 5-fold decrease at 2 hours. The urine and tissue levels of interleukin (IL)-1β, IL-4, and growth-related oncogene/keratinocyte-derived chemokine correlated significantly, with a positive Spearman correlation also noted for granulocyte-macrophage colony-stimulating factor, monocyte chemotactic protein-1-1, IL-18, and interferon-γ. The tissue levels for most cytokines, except for IL-2, and urinary frequency were significantly elevated in the CYP-treated rats compared with the control vehicle-treated rats. The hints of severe inflammation in the bladder indicated by the urinary cytokines were confirmed by bladder histologic examination and the tissue cytokine levels at necropsy. Conclusions: The progression of CYP-induced cystitis was clearly reflected in the urine matrix by the temporal and quantitative changes in the cytokine levels. Additional delineation of urine and bladder tissue cytokine expression might yield biomarkers for cystitis.