Background: Although feline urine is increasingly submitted for bacterial culture and susceptibility testing in veterinary practice, bacterial cystitis (BC) is relatively uncommon compared with feline interstitial cystitis (FIC), which shares similar clinical manifestations. Therefore, an investigation of certain urothelial (glycosaminoglycan [GAG], tissue inhibition metalloproteinase-2 [TIMP-2]), cytokine (interleukin 12 [IL-12]), and neurotrophic factor (nerve growth factor [NGF]) markers may aid diagnosis. Objectives: We aimed to evaluate the diagnostic effectiveness of selected serum/urine biomarkers in the diagnosis of cats with FIC and BC. Methods: Twelve healthy cats (Control group) and 24 cats with feline lower urinary tract disease (FLUTD) were used, and the cats with FLUTD were divided into FIC and BC groups. Results: When comparing the three groups, serum GAG, IL-12, NGF, and TIMP-2 concentrations were highest in the FIC group; urine GAG, IL-12, NGF, and TIMP-2 concentrations were higher in the FIC and BC groups than those in the Control group. Serum NGF concentrations were higher in the FIC group than in all other groups. Also, serum GAG, IL-12, NGF, and TIMP-2 concentrations were found to be effective in the differential diagnosis of FIC vs BC. Conclusions: We showed that serum NGF is a candidate biomarker that could be used in the diagnosis and differentiation of FIC. Urine GAG, IL-12, NGF, and TIMP-2 concentrations might be helpful in determining urinary bladder inflammation and/or damage in cats with FIC and BC. ROC analyses revealed that serum and urine biomarkers were effective for diagnosing FIC and that serum biomarkers rather than urine biomarkers were effective for the differential diagnosis of FIC and feline BC.