Our objective was to determine whether serial HIV testing during pregnancy and the postpartum period as well as male partner testing are acceptable and feasible in Tororo, Uganda. This was a prospective study of pregnant women at the Tororo District Hospital (TDH) Antenatal Clinic. Patients presenting for routine antenatal care were asked to participate in a serial HIV testing integrated into standard antenatal and postpartum/child immunization visits, and to invite their male partners for HIV testing. Serial testing was defined as ≥2 tests during pregnancy and ≥2 tests within 24 weeks postpartum. Of the 214 enrolled women, 80 (37%) completed serial testing, 176 (82%) had ≥2 tests, and 147 (69%) had ≥3 tests during the study period. One hundred eighty-two women (85%) accepted male partner testing, but only 19 men (10%) participated. One woman seroconverted during the study, for a cumulative HIV incidence of 0.5% (1/214). In multivariable logistic regression analysis, longer distance between home and clinic (aOR 0.87 [95% CI 0.79-0.97]) and not knowing household income (aOR 0.30 [95% CI 0.11-0.84]) were predictive of not completing serial testing. Higher level of education was associated with completing serial testing (linear trend p value = 0.05). In conclusion, partial serial HIV testing was highly acceptable and feasible, but completion of serial testing and male partner testing had poor uptake.