P-glycoprotein (P-gp), a transporter membrane protein, plays an important role in various physiological and physiopathological conditions, drug-drug and drug-food interactions, and multi-drug resistance. Nowadays, P-gp modulators are used consciously to treat some specific diseases, such as cancer and poisoning. Levofloxacin (LVX), a P-gp substrate, may cause the stimulation of the central nervous system. The aim of this study was to determine the effects of fexofenadine (FEX) and dexamethasone (DEX) with P-gp modulators on the distribution of LVX in rat brain, testicle, and plasma. In the in vivo section of the study, LVX concentrations in tissue and plasma samples were detected by high-performance liquid chromatography. In addition, in silico molecular docking studies were conducted to elucidate interactions between P-gp and drugs. The administration of DEX at a low dose and minute 30 reduced the LVX passage to the brain, while the administration of DEX at a high dose and hour 2 increased the LVX passage to the brain, testicle tissue, and plasma. MM-GBSA ΔG values of FEX, DEX, and LVX were calculated to be−80.95,−57.74, and−52.12 kcal/mol, respectively. The results of this study reported that DEX, known as a P-gp inductor, may show inhibitory activity based on dose, tissue, and time of application.