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Introduction The Applied Health Research Question (AHRQ) portfolio is an initiative funded by the Ontario Ministry of Health. Knowledge users submit AHRQ requests to use administrative health data to inform their planning, policy and program development. Objectives and Approach A request to estimate prevalence and incidence of blood-borne viral infections (BBVI) among individuals seeking fertility treatment services was approved by the ICES AHRQ team. To determine whether current BBVI testing guidelines are effective for timely identification of BBVI among individuals undergoing fertility treatment, this AHRQ sought to estimate: a) the testing prevalence for BBVI (using viral hepatitis as a proxy for all BBVI including HIV), and b) BBVI incidence over a five-year follow-up period. We used infertility codes billed by gynecologists and urologists to identify individuals seeking fertility treatment between April 1, 2002 and March 31, 2013. We looked forward five years from a first BBVI test billed by a physician and assessed annual and cumulative testing prevalence and incidence of viral hepatitis and HIV using rule-based administrative algorithms. Results In total, 184,328 individuals (61.6% female) were included. The five-year BBVI testing prevalence was 45.5% and the median number of tests was 2 (interquartile range: 2-3). An estimated 949 new cases (48% diagnosed within 12 months of first BBVI test) of viral hepatitis were identified in the five-year follow-up with a cumulative incidence of 522 (95%CI 489-556) per 100,000 population. There were 57 new HIV cases (68% diagnosed within 12 months of first BBVI test) with a cumulative incidence of 31.4 (95%CI 23.7-40.6) per 100,000. The median times from first BBVI test to hepatitis and HIV diagnoses were 13.0 months (IQR 2.33-32.2) and 3.0 months (IQR 1.34-20.0), respectively. Conclusion / Implications These findings can be used to develop evidence-based BBVI testing guidelines for individuals seeking fertility treatment services.