In situ gel-forming systems have drawn much attention of current researchers to overcome the poor bioavailability from the conventional eye drops. The present work described formulation and pharmacoscintigraphic evaluation of timolol-maleate-loaded chitosan/hydroxy propyl methyl cellulose (HPMC)-based polymer matrix for enhanced ocular retention. Chitosan and HPMC ratio was optimized and formulation was characterized for various in vitro parameters. The ocular retention was studied on New Zealand rabbits by gamma scintigraphy, which is a very simple and noninvasive technique. For scintigraphy study, the drug timolol maleate was radiolabeled (99m)Tc by direct labeling method using SnCl(2).2H(2)O as reducing agent. The labeling procedure was optimized to get maximum labeling efficiency (>98%). In vitro stability of the radiolabeled drug ((99m)Tc-timolol maleate complex) was checked and it was found to be stable for up to 24 h. Plain drug eliminates rapidly as significant activity was recorded in kidney and bladder after 2 h of ocular administration. It was evident from the scintigraphic images and the time-activity curve plotted from the data that the plain drug solution cleared very rapidly from the corneal region and reached into systemic circulation via nasolachrymal drainage system, as significant activity was recorded in kidney and bladder after 2 h of ocular administration. Developed formulation cleared at a slow rate and remained at corneal surface for longer time duration. No radioactivity was observed in systemic circulation after 2 h. Ocular irritation of the developed formulation was also checked by hen's egg chorioallantoic membrane test and formulation was found to be practically nonirritant. The study signified the potential of gamma scintigraphy in evaluation of novel drug delivery systems in a noninvasive manner.