The aim of this study was to validate City of Hope Quality of Life-Ostomy Questionnaire (CoH-QoL-OQ) for assessing the quality of life (QoL) of ostomy patients in the Republic of Croatia. The CoH-QoL-OQ is widely used, but has not been translated or validated so it can be used in the Republic of Croatia. This cross-sectional study encompassed 302 surgery patients with colostomy, ileostomy, or urostomy (182 (60.3%) male and 120 (39.7%) female), whose average age is 59 (M = 59.3, SD = 15.8). The CoH-QoL-OQ was translated into Croatian language using accepted guidelines for translation. Patients were recruited in a telephone conversation, followed by mail containing the CoH-QoL–OQ delivered to the home addresses of the patients who agreed to participate. The collected data were analyzed to verify psychometric properties of the questionnaire on the Croatian sample. All subscales showed high level of internal consistency (Cronbach α = 0.73–0.89). The test-retest reliability indicated a very satisfactory temporal stability (r = 0.99). The Confirmatory Factor Analysis (CFA), showed that the originally established model was not adequate for the data (χ2 = 4237.88, p < 0.01, CFI = 0.540, NNFI = 0.481, RMSEA = 0.113). However, after modification that excluded problematic items, the data showed a better fit with the theoretical model (except for the LR chi-square test that remained statistically significant: χ2 = 1144.28, p < 0.01, CFI = 0.869, NNFI = 0.855 RMSEA = 0.077). We conclude that the CoH-QoL-OQ is a valid, reliable, and reducible instrument for measuring the health-related quality of life (HRQoL) among Croatian patients with ostomy in clinical research and clinical practice.