This reference manual, published by PEPFAR and the U.S. Centers for Disease Control and Prevention, is a guide for Implementation Subject Matter Experts (ISME)s who offer either in country or remote technical assistance in viral load (VL) and early infant diagnosis (EID) scale-up. Collectively, it presents best practices and proposed solutions for the challenges that are common across PEPFAR programs. This manual can help guide ISMEs as they work with countries on challenges that are beyond their area of expertise. This is a living document and can help support the efforts of ISMEs and field colleagues to implement high quality, routine VL and EID testing for all patients on antiretroviral therapy.