Mother-to-child transmission (MTCT), or ‘vertical transmission’ is a significant contributor to the acquisition of HIV with an estimated 150 000 new HIV infections among children reported in 2020, and other infectious diseases including syphilis and hepatitis B (HBV). The 2021 World Health Organization global guidance on criteria and processes for validation: elimination of mother-to-child transmission of HIV, syphilis and hepatitis B virus is an update of the second edition published in 2017. This version (3rd ed) provides standardised processes and consensus-developed criteria to validate MTCT of HIV, syphilis and HBV, and recognises high-burden countries that have made significant progress on the path to elimination. Additionally, it emphasises country-led accountability, rigorous analysis, intensive program assessment and multi-level collaboration, including the involvement of communities of women living with HIV and/or HBV. Foundational requirements are reviewed in the four areas of (i) data, (ii) laboratory, (iii) program and (iv) human rights, gender equality and community engagement. This guide is of high utility to stakeholders involved in planning, implementing and monitoring family health programs at international, regional and national levels.