The present study involves the use of mixed cosolvency solubilisation technique to estimate the amount of Acyclovir present in tablets by spectrophotometric method in the mixture of solvents 15% urea, 8% propylene glycol and 7% PEG 6000. It was observed that solubility of Acyclovir in this solvent mixture was increased by ten folds as compared with distilled water. The method obeyed Beer’s law with maximum absorbance at 251nm. The analysis of tablets indicated good correlation between estimated and label claim. The results of recovery study revealed that any small change in the drug concentration in the solution could be accurately determined by the proposed method. The low values of LOD and LOQ of Acyclovir in the solvent mixture indicated good sensitivity of proposed method. As urea, propylene glycol and PEG 6000 were cheaper than most of the organic solvents, these can be used as a substitute for organic solvents. The study proved that mixed cosolvency phenomenon is an effective technique in enhancement of aqueous solubility of poorly water soluble drugs. The proposed method is new, simple, accurate, non-toxic and precise method that can be successfully employed for estimation of drugs in routine analysis of tablets.