The aim of the present prospective study was to assess the diagnostic benefit of UroVysion (Vysis-Abbott Laboratories, Downers Grove, IL) in the follow-up of patients with a history of high-grade non-muscle-invasive urothelial carcinoma of the bladder (NMIBC). An unselected cohort of 25 patients with a history of high-grade NMIBC was prospectively followed up by office-based cystoscopy, cytology, and UroVysion in 210 events. The sensitivity and specificity for standard combined cystoscopy and cytology were 78% and 83%, respectively. UroVysion yielded a considerably higher detection rate with 94% and 93%, respectively. In 89% of the follow-up events of patients with a history of previous carcinoma in situ (CIS) and negative cystoscopy but a positive UroVysion finding, CIS recurrence was noticed within 5 months. UroVysion is a worthwhile approach in patients with previous CIS, a high risk for the development of CIS, or previous unequivocal cytology suggestive of CIS, especially during or shortly after instillation therapy.