This study evaluates the influence of pregnancy and HIV infection in conjunction with the use of raltegravir, lamivudine, and tenofovir disoproxil fumarate (combined antiretroviral therapy [cART]) on intestinal P‐glycoprotein (P‐gp) and hepatic organic anion transporter polypeptide (OATP) 1B1/1B3 and/or breast cancer resistance protein (BCRP) drug transporter activity using rosuvastatin (OATP1B/BCRP) and fexofenadine (P‐gp) probes. Single oral doses of 5‐mg rosuvastatin and 60‐mg fexofenadine were administered to women living with HIV under cART in the third trimester of gestation (n = 15) and postpartum period (n = 10). A control group of 12 healthy nonpregnant women also was investigated. Pharmacokinetic parameters were estimated by using a noncompartmental method and evaluated by t test (P < .05). The rosuvastatin area under the plasma concentration–time curve from time 0 to the last quantifiable concentration (AUC0‐last) value was higher in the third trimester of pregnancy (19.5 [95%CI, 16.8–22.3] ng • h/mL] when compared to postpartum (13.3 [95%CI, 9.3–17.5] ng • h/mL), while the fexofenadine AUC0‐last values did not differ between the third trimester of pregnancy (738.0 [95%CI, 611.4–864.6] ng • h/mL) and postpartum period (874.9 [95%CI, 408.2–1342.0] ng• h/mL). The rosuvastatin AUC0‐last values did not differ between healthy nonpregnant women (13.8 [95%CI, 10.0–17.6] ng • h/mL) and women living with HIV in the postpartum period (13.3 [95%CI, 9.3–17.5] ng • h/mL), and the fexofenadine AUC0‐last values did not differ between the 2 investigated groups (603.6 [95%CI, 467.5–739.7] ng • h/mL vs 874.9 [95%CI, 408.2–1342.0] ng • h/mL). It is suggested that gestation inhibits the hepatic OATP1B1/1B3 and/or BCRP activity but does not alter intestinal P‐gp activity. The influence of HIV infection in conjunction with use of cART on OATP1B/BCRP and intestinal P‐gp activity was not observed.