International initiatives to strengthen national health laboratory systems in resource-poor countries are often hampered by unfamiliarity with the country's health laboratory environment and turnover of international partners during the initiative. This study provides an overview of, and lessons learned from, the use of a laboratory long-term partnership approach (ie, "twinning") to strengthen the national public health laboratory system in an international setting. We focused on the partnering of the Uganda Ministry of Health Central Public Health Laboratory (CPHL) with the New Mexico State Public Health Laboratory to help the CPHL become Uganda's national public health reference laboratory (Uganda National Health Laboratory Services [UNHLS] Institute) and leader of its nascent Uganda National Health Laboratory Network (UNHLN). Via twinning, CPHL leadership received training on laboratory leadership and management, quality systems, facility management, and the One Health environmental strategy (ie, that the health of persons is connected to the health of animals and the environment), and drafted a National Health Laboratory Policy, UNHLS Institute business plan, and strategic and operating plans for the UNHLS Institute and UNHLN. The CPHL is now responsible for the UNHLS Institute and coordinates the UNHLN. Lessons learned include (1) twinning establishes stable long-term collaborations and (2) success requires commitment to a formal statement of activities and objectives, as well as clear and regular communication among partners.