The prodrug strategy has been frequently employed as a chemical approach for overcoming the disadvantages of existing parent drugs. In this report， we synthesized four monoester prodrugs of ganciclovir， an anticytomegalovirus drug， and demonstrated their potential advantages in protease-targeted activation and biopharmaceutical profiles over the parent compound. We demonstrated that these four prodrugs of ganciclovir， i.e.， N-benzyloxycarbonyl-(L)-alanine-ganciclovir (CbzAlaGCV)， N-benzyloxycarbonyl-(α，l)-aminobutyric acid-ganciclovir (CbzAbuGCV)， N-acetyl-(l)-phenylalanine-(l)-alanine-ganciclovir (AcPheAlaGCV)， and N-acetyl-(l)-phenylalanine-(α，l)-aminobutyric acid-ganciclovir (AcPheAbuGCV)， are hydrolytically activated by the protease of human cytomegalovirus (hCMV)， a serine protease that possesses intrinsic esterase activities. CbzAlaGCV and AcPheAlaGCV were found to be activated at a higher rate by the hCMV protease than CbzAbuGCV and AcPheAbuGCV. These ganciclovir prodrugs could potentially be targeted to selective activation by the hCMV protease which is only present at the viral infection sites， thereby achieving higher efficacy and lower systemic toxicity. The tissue stability， cellular uptake， and trans-epithelial transport of these ganciclovir prodrugs were also characterized. The N-acetylated dipeptide prodrugs of ganciclovir were found to be generally more stable than Cbz-amino acid prodrugs in various tissue matrices. Among the four prodrug candidates， AcPheAbuGCV was the most stable in human cell homogenates， plasma， and pooled liver microsomes. AcPheAbuGCV also possessed a superior cellular uptake profile and permeability across epithelial cell monolayers. Since the targeting and selective activation of a prodrug is determined by not only its rate of hydrolysis catalyzed by the hCMV protease target but also its biopharmaceutical properties， i.e.， oral absorption and systemic availability， AcPheAbuGCV is considered the best overall candidate among the four ganciclovir prodrugs for further research and development for treatment of hCMV infection.