A drug-drug interaction (DDI) trial of cytochrome P450 3A (CYP3A) is a necessary part of early-phase trials of drugs mainly metabolized by this enzyme, but CYP3A DDI clinical trials do not have a standard design, especially for Chinese people. We aimed to offer specific recommendations for CYP3A DDI clinical trial design. This was an open, three-cycle, self-controlled study. Healthy subjects were given different administration strategies of CYP3A4 perpetrators. In each cycle, blood samples were collected before and within 24 h after the administration of midazolam, the CYP3A indicator substrate. The plasma concentrationsof midazolam and 1-hydroxymidazolam was obtained using liquid chromatography tandem mass spectrometry assay. For CYP3A inhibition, itraconazole exposure with a loading dose could increase the exposure of midazolam by 3.21-fold based on maximum plasma concentration (Cmax), 8.37-fold based on area under the curve Pharmacology Research & Perspectives for review only from zero to the time point (AUC0-t), and 11.22-fold based on area under the curve from zero to infinity (AUC0-∞). The data were similar for itraconazole pretreatment without a loading dose. For CYP3A induction, the exposure of rifampin for 7 days decreased the plasma concentration of midazolam ~0.27-fold based on Cmax, ~0.18-fold based on AUC0-t, and ~0.18-fold based on AUC0-∞. Midazolam exposure did not significantly change when the pretreatment of rifampin increased to 14 days. This study showed that itraconazole pretreatment for 3 days without a loading dose was enough for CYP3A inhibition, and pretreatment with rifampin for 7 days could induce near-maximal CYP3A levels.