Acute Cystitis Symptom Score (ACSS) is an 18-item self-reporting questionnaire for clinical diagnosis and follow-up of acute uncomplicated cystitis (AUC) in women. The ACSS, originally developed in Uzbek and Russian languages, is now available in several languages. The purpose of the study was to validate the ACSS questionnaire in the Italian language. Linguistic validation was carried out according to Linguistic Validation Manual for Patient-Reported Outcomes Instruments guidelines. Clinical validation was carried out by enrolling one hundred Italian-speaking women. All women were asked to fill in the ACSS questionnaire during their medical visit. Fifty-four women, median age 36 (Inter Quartile Range 28–49), were diagnosed with AUC, while 46 women, median age 38 (IQR 29–45), were enrolled as the control group attending the hospital’s fertility center for couples. The most frequently isolated pathogen in AUC was Escherichia coli (40; 74.0%) followed by Enterococcus faecalis (7; 13.0%) and Staphylococcus saprophyticus (3; 5.6%). Receiver operating characteristic (ROC) curve analysis performed at the first diagnostic visit on a typical symptoms domain cut-off score of 6 revealed a sensitivity of 92.5% and specificity of 97.8%. The Italian version of the ACSS has proved to be a reliable tool with a high accuracy in diagnosis and follow-up in women with AUC. The ACSS may also be useful for clinical and epidemiological studies.