Trimethoprim–sulfamethoxazole (TMP–SMX) prophylaxis in HIV-uninfected, exposed (HUE) children variably reduces clinical malaria burden despite antifolate resistance, but data regarding achieved serum levels and adherence are lacking. Serum samples from 70 HUE children aged 3–12 months from Rakai, Uganda, enrolled in an observational study were assayed for random SMX levels using a colorimetric assay. Adherence with TMP–SMX prophylaxis data (yes/no) was also collected. Of 148 visits with concurrent SMX levels available, 56% had self-reported adherence with TMP–SMX therapy. Among these 82 visits, mean (standard deviation) level was 19.78 (19.22) µg/mL, but 33% had SMX levels below half maximal inhibitory concentrations (IC50) for Plasmodium falciparum with some, but not all, of the reported antifolate resistance mutations reported in Uganda. With TMP–SMX prophylaxis, suboptimal adherence is concerning. Sulfamethoxazole levels below IC50s required to overcome malaria parasites with multiple antifolate resistance mutations may be significant. Further study of TMP–SMX in this context is needed.