Laboratory investigations are vital in preventive, diagnostic, therapeutic, and referral services. Efficient delivery of medical laboratory testing services at all levels and locations of healthcare services, especially in rural and poor populations, is essential. Nigeria’s diagnostic landscape was plagued with underutilization of medical laboratory equipment, and a formal structure for referral and transport of diagnostic specimens to facilitate rapid diagnosis and laboratory confirmation needed to be revised. To provide quality medical laboratory testing services equitably and cost-effectively, Nigeria needed an integrated, multi-level specimen referral system. The National Integrated Specimen Referral Network (NiSRN) became operational in March 2018. This study examines the NiSRN operations’ impact on attaining the third 95 of UNAIDS 95-95-95 targets. Descriptive methodologies were used to assess the viral load (HIV-VL) testing in Nigeria before NiSRN began. Between 2018 and 2022, annual volumes of VL and EID samples transported across the 36 states and the Federal Capital Territory (FCT) and assayed by the Polymerase Chain Reaction (PCR) laboratories were measured. VL suppression was reviewed. Using NiSRN, specimen backlogs were identified and relocated to laboratories that could promptly be analyzed. Eight hundred ninety-nine thousand nine hundred twelve specimens were transported in 2019, rising to 2,001,184 in 2022. VL coverage was 55% in 2018 and steadily rose to 95% in 2022. VL suppression rate was 78% in 2020 and rose to 96% in 2022, exceeding the UNAIDS’ third 95 target. The NiSRN, operated by the Federal Government of Nigeria with support from PEPFAR and the Global Fund, has helped to exceed the UNAIDS’ third 95 target. As evidenced by these results, PEPFAR and Global Fund teams in Nigeria should continue to support the NiSRN. Nigeria’s Government must devise means of continuous sustainability with innovations. Other countries struggling to meet the UNAIDS third 95 can learn from Nigeria’s experience.