Mecobalamin is one of the active forms of vitamin B12 used in treatment of megaloblastic anemia, peripheral neuropathy and also in pernicious anemia. The main objective of that experimental work was to develop an analytical method for the validation of mecobalamin by UV/Vis Spectrophotometer (Shimadzu). Different analytical parameters were validated by using pH-7.0 buffer (monobasic potassium Dihydrogen phosphate) as a solvent for mecobalamin injection. All the results were found within the limited range as prescribed by ICH guidelines. The stable absorbance maxima for mecobalamin was found at 522nm by using pH-7.0 buffer scanning under the range of 200-600nm. The performed procedure was linear within solution strength range of 16μg/mL to 24μg/mL with correlation coefficient (r2=0.9987). The studies of recovery and precision were performed and results were observed in specified range as per ICH guidelines. The % RSD for precision and intermediate precision were found 0.004326% and 0.004326-0.004333% respectively. In accuracy mean % recovery was calculated as 99.90-100.0% proved that the focused method was accurate and according to all sequels, it is applicable for routine analysis of mecobalamin in injection form in Pharma-industries.