This research examined the effect of the active fragment of the absorption enhancer, Zonula occludens toxin (Zot), DeltaG, and potential P-glycoprotein (P-gp) inhibitors, the enaminones, on transepithelial transport and oral bioavailability of paracellular markers and/or low bioavailable drugs. The first approach focused on studies performed with the newly isolated biologically active fragment of Zot, DeltaG. The permeability of paracellular markers and low oral bioavailable compounds was studied in Caco-2 cells with/without DeltaG accompanied with cytotoxicity studies. In addition, DeltaG effect on the oral absorption of paracellular markers and low bioavailable drug candidates was investigated in jugular vein cannulated Sprague Dawley rats via intraduodenal administration. The second approach focused on a novel class of chemical compounds, the enaminones, as potential P-gp inhibitors. In vitro screening studies by efflux inhibition identified potential P-gp inhibitors, DM40 and DM27. The in vitro transport, in vivo absorption, and brain distribution were studied for P-gp substrates with DM27 and DM40. The results of this research displayed the effectiveness of AG in improving the in vitro and in vivo transport of paracellular markers and low bioavailable drugs. The in vitro transport enhancement reached 300% without any toxicity. Despite susceptibility to metabolism, DeltaG produced significant increases in Cmax and AUC, for all the investigated drugs with protease inhibitors (PI), up to 57 and 50 fold, respectively, for cyclosporin A. For certain agents, the increase in drug absorption observed with DeltaG/PI was a composite effect of DeltaG modulation of tight junctions and PI inhibition of metabolism. The screening of the enaminones revealed two P-gp inhibitors, DM27 and DM40. DM27 was more potent than DM40. Despite the distinctive enhancement observed with paclitaxel/DM27, further in vitro evaluation of the effect of DM27 with P-gp substrates displayed significant transport increases within ∼23 to 51% without any toxicity or effects on the paracellular/transcellular routes. In vivo studies found that both enaminones increased the brain levels of paclitaxel with a maximum of 227%. In conclusion, this research displayed the potential usefulness of paracellular absorption modulation by DeltaG and efflux inhibition by enaminones to increase the transport and/or oral bioavailability of therapeutic drugs.