Background: Although bacterial cystitis (BC) and feline interstitial cystitis (FIC) are categorized under feline lower urinary tract disease (FLUTD) due to their similar clinical manifestations, stress is an important factor for FIC. Therefore, the investigation of stress biomarkers might be important in the differentiation and elucidation of these conditions. Objectives: We aimed to evaluate the diagnostic effectiveness of serum and urine cortisol, serotonin, and dopamine concentrations and their relationship with stress in cats with FIC and BC. Methods: Twelve healthy cats (Control group) and 24 cats with FLUTD were used. The cats with FLUTD were divided into FIC and BC groups. Results: Multimodal environmental modification (MEMO) scores were found to be higher in the FIC group than in the BC and Control groups (P <.001). Urine serotonin concentrations were higher in cats with FIC and BC compared with those in the Control group. Based on ROC analyses, the sensitivity, specificity, and accuracy of urine serotonin and dopamine were found to be statistically significant in being able to differentially diagnose cats in the FIC group vs the Control group. The sensitivity, specificity, and accuracy of serum dopamine were also found to be statistically significant for the differential diagnosis of FIC and BC. Conclusions: High urine serotonin concentrations were found in cats with FLUTD compared with healthy controls and interpreted as the presence of stress not only in cats with FIC but also in cats with BC. Also, based on the ROC-based diagnostic performance evaluation of these stress biomarkers, urine serotonin, and dopamine concentrations can be used to diagnose FIC, and serum dopamine concentrations can be used to differentiate FIC and BC in cats.