This study aimed to determine the association between the plasma concentration of nevirapine (NVP) and clinical outcomes. In this cross-sectional study, sociodemographic and clinical data were collected from 233 HIV patients receiving NVP-based first-line antiretroviral therapy (ART) regimens in Nairobi, Kenya. The mean age was 41.2 (SD ± 11.9) years. Fifty-four (23.2%) patients had virological failure (>1000 copies/mL), whereas 23 (9.9%) were infected with drug-resistant HIV strains. Eleven patients had nucleoside reverse transcriptase inhibitor resistance mutations, including M184V and T215Y, whereas 22 had non-nucleoside reverse transcriptase inhibitor resistance mutations, including G190A, K103N, V106A, Y181C, A98G, and Y188L. The median NVP plasma concentration was 6180 ng/mL (IQR 4444–8843 ng/mL), with 38 (16.3%) patients having suboptimal NVP plasma levels of <3400 ng/mL. The majority 23 of the 38 (60.5%) patients with NVP Cmin < 3400 ng/mL were significantly infected with drug-resistant HIV virus (P = .001). In the multivariate analysis, the time taken to arrive at the ART clinic (β −11.1, 95% CI −21.2 to −1.1; P = .031), higher HIV viral load (β −2008, 95% CI −3370.7 to −645.3; P = .004), and the presence of HIV drug resistance mutation (β 3559, 95% CI 2580.8–4537.2; P = .0001) were associated with NVP plasma concentration. A significant proportion of patients receiving the NVP-based regimen had supra- and sub-therapeutic plasma concentrations. Higher HIV viral load and the presence of HIV drug-resistant mutations are important factors associated with NVP plasma concentrations.