Valganciclovir HCl (VGH) is the widely used drug for the treatment of cytomegalovirus (CMV) retinitis infection with an induction dose of 900mg per oral (p.o.) twice a day and a maintenance dose of 900mg (p.o.). This required dose of the drug also leads to multiple side effects due to repeated administration. The research was highlighted to develop, formulate, optimize, and evaluate single-core osmotic pump (SCOP) tablet of VGH with the dose of 450mg to reduce dosing frequency and associated side effects. The decrease in dose also minimizes the hepatic and nephrotic load. The optimized batch of the formulation was subjected to comparative in vitro and in vivo evaluation. The tablet core composition is the primary influencer of the drug delivery fraction in a zero order, whereas the membrane characteristics control the drug release rate. In vivo pharmacokinetic studies revealed that the newly developed osmotic formulation has controlled zero-order release for 24h with a single dose of 450mg while the marketed formulation requires twice administration within 24h to maintain the plasma concentration in the therapeutic window. The pharmacokinetic study demonstrated that the developed formulation has the area under curve (AUC) of 58.415µgh/ml with single dose while the marketed formulation shows the AUC of about 37.903µgh/ml and 31.983µgh/ml for first and second dose, respectively. The large AUC demonstrates the extended release of drug with a single dose and effective plasma concentration. Hence, the developed formulation can be a promising option for the treatment of CMV retinitis with the minimum dose and dosing frequency.