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Background: Passive surveillance is the principal method of sexually transmitted infection (STI) and blood-borne virus (BBV) surveillance in Australia whereby positive cases of select STIs and BBVs are notified to the state and territory health departments. A major limitation of passive surveillance is that it only collects information on positive cases and notifications are heavily dependent on testing patterns. Denominator testing data are important in the interpretation of notifications. Objective: The aim of this study is to establish a national pathology laboratory surveillance system, part of a larger national sentinel surveillance system called ACCESS (the Australian Collaboration for Coordinated Enhanced Sentinel Surveillance). ACCESS is designed to utilize denominator testing data to understand trends in case reporting and monitor the uptake and outcomes of testing for STIs and BBVs. Methods: ACCESS involves a range of clinical sites and pathology laboratories, each with a separate method of recruitment, data extraction, and data processing. This paper includes pathology laboratory sites only. First established in 2007 for chlamydia only, ACCESS expanded in 2012 to capture all diagnostic and clinical monitoring tests for STIs and BBVs, initially from pathology laboratories in New South Wales and Victoria, Australia, to at least one public and one private pathology laboratory in all Australian states and territories in 2016. The pathology laboratory sentinel surveillance system incorporates a longitudinal cohort design whereby all diagnostic and clinical monitoring tests for STIs and BBVs are collated from participating pathology laboratories in a line-listed format. An anonymous, unique identifier will be created to link patient data within and between participating pathology laboratory databases and to clinical services databases. Using electronically extracted, line-listed data, several indicators for each STI and BBV can be calculated, including the number of tests, unique number of individuals tested and retested, test yield, positivity, and incidence. Results: To date, over 20 million STI and BBV laboratory test records have been extracted for analysis for surveillance monitoring nationally. Recruitment of laboratories is ongoing to ensure appropriate coverage for each state and territory; reporting of indicators will occur in 2019 with publication to follow. Conclusions: The ACCESS pathology laboratory sentinel surveillance network is a unique surveillance system that collects data on diagnostic testing, management, and care for and of STIs and BBVs. It complements the ACCESS clinical network and enhances Australia’s capacity to respond to STIs and BBVs. International Registered Report Identifier (IRRID): RR1-10.2196/13625