The objective of the research work was focused on optimization and evaluation of Mesalamine rectal in-situ gel of controlled/sustained release. This is used as anti-ulcerative colitis drug, for pediatric dose.The in-situ gel formulation were prepared by simple mixing method it's a cooling technique based on the temperature triggering system by incorporation of various polymers like Poloxamer 407 and HPMC E-50 in different proportions. The in-situ gel characteristics were evaluated for clarity, gelation time, gelation temperature, gel strength, and in-vitro drug release studies. The formulations gave an initial burst effect and followed by sustained release for 12 h. Optimized formulation showed release of drug up to 92.860% in 12 h. Optimization was done by using design expert software. The optimized formula (F3) showed no significant changes on stability studies when stored at 40ºC/75% RH for one month according to ICH guidelines.