The Acute Cystitis Symptom Score (ACSS) is a patient self-reporting questionnaire for the clinical diagnosis and patient-reported outcome (PRO) in women with acute uncomplicated cystitis (AC). The aim of the current study (part II) was the clinical validation of the Greek ACSS questionnaire. After linguistic validation according to internationally accepted guidelines and cognitive assessment (part I), the clinical validation was performed after ethical approval by using the Greek ACSS study version in 92 evaluable female participants including 53 patients with symptoms suspicious of AC and 39 controls. The clinical outcome using the ACSS questionnaire at different time points after the start of treatment was demonstrated as well. The age (mean±SD) of the 53 patients (44.7±17.0) and 39 controls (49.3±15.9) and their additional conditions at baseline visits, such as menstruation, premenstrual syndrome, pregnancy, menopause, diabetes mellitus, were comparable. There was, however, a significant difference (p<0.001) between patients and controls at baseline visit regarding sum score of the ACSS domains, such as typical symptoms and quality of life. The clinical outcome of up to 7 days showed a fast reduction of the symptom scores and improvement of quality of life. The optimal thresholds for the patient-reported outcome of successful therapy could be established. The linguistically and clinically validated Greek ACSS questionnaire can now be used for clinical or epidemiological studies and also for patient’s self-diagnosis of AC and as a PRO measure tool.