N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) is the peptidoglycans obtained from the Mycobacterium tuberculosis. The isolate Mycobacterium tuberculosis (MT1) was obtained from the sputum of the tuberculosis patients. The MT1 isolates undergone for the morphological, biochemical and staining technique for the conformation of Mycobacterium tuberculosis. The mid log phase MT1 isolates were used for the extraction of MDP by using HPLC. The muropeptides were eluted from the HPLC with 2 to 30% linear gradient of aceto nitrile containing 0.5% formic acid at 320 L/min. The purified MDP was eluted with in the retention time of standard Muramyldipeptide.