Antiretroviral therapy (ART) is a cornerstone of HIV management, and viral load testing serves as the gold standard for assessing drug effectiveness and confirming treatment adherence. The objectives set by The Joint United Nations Programme on HIV/AIDS (UNAIDS) for HIV care and treatment still encounter challenges, particularly in achieving improved access to ART and associated services, including essential laboratory viral load monitoring. This study investigated gaps in routine viral load monitoring within the 95-95-95 cascade, with particular emphasis on the final 95%, which remains unattained across individual, programmatic, and population levels. The research adopted a cross-sectional survey design with a pragmatic approach, focusing on healthcare professionals providing HIV services in clinics in Nairobi. A proportional sample of 226 respondents was collected from a population of 550 to gather qualitative and quantitative data. The study yielded a 90% response rate, with 96% agreement among respondents. The findings revealed a moderately low but statistically significant positive correlation (r=0.479<0.05). The R2 value of 0.230 explained 23.3% of project performance and displayed statistical significance (p-value=0.000<0.05). Consequently, the null hypothesis was rejected, indicating a substantial relationship between routine viral load monitoring services and the performance of HIV projects. In light of these results, the study recommends the implementation of a combination of feasible strategies, tailored to differentiated care models, to address challenges in low-resource settings across various population groups. These strategies aim to enhance adherence and improve viral load suppression rates, thus promoting the objectives of HIV care and treatment.