Till date, pursuit for cost effective and animal sparing colon specific bio-relevant dissolution media has been a foremost challenge facing pharmaceutical scientists over many decades.It is problematic to mimic the dynamic and ecologically diverse features of the colon in dissolution vessel.With the knowledge of enormous colonic microflora, the predominant species Bacteroides, Bifidobacterium, Eubacterium, Streptococcus and Lactobacillus species were cultured in 12% w/v skimmed milk powder and 5%w/v grade “A” honey. Probiotic culture was added to the dissolution media in order to test the drug release of polysaccharide based formulations. USP dissolution apparatus I/II with gradient pH dissolution method were used to evaluate the drug release from formulations meant for colonic drug delivery. Drug release from 5-fluorouracil granules and metronidazole tablets were assed under gastric, small intestine conditions and also within a simulated colonic environment involving existing rat caecal, human fecal media and compared with novel probiotic media. The present method can be successfully applied for the drug release testing of any oral formulations meant for colonic delivery.