Purpose:Vitamin B12 plays vital role in nervous system functioning, in the formation of RBC, and in the synthesis of DNA. Vitamin B12 deficiency will result in irreversible damage to the nervous system. To understand Vitamin B12 deficiency and related diseases, it is required to know its concentrations where a direct, selective and sensitive quantification analytical method is required. Currently, vitamin B12 is quantified by microbiological and chemical assays which are indirect and non-specific. Till date there is no report on direct mass spectrometry method to quantify Vitamin B12 concentrations from biological matrices. Here, we present a sensitive LC-MS/MS method for quantification of Vitamin B12 in serum which is applied to pharmacokinetic study. Method:100 µL of serum sample was taken into 2 mL tubes and 10µL of internal standard was added followed by 1mL of ice cold 10% formic acid in water. SPE sample extraction was performed using Waters HLB® cartridges. Samples were loaded on to previously equilibrated cartridge and washed with 3 mL of water followed by 1 mL of 5% methanol. Samples were eluted with 200µL of 10 mM ammonium formate in 0.2% formic acid v/v and 0.2% ascorbic acid, w/v and acetonitrile (75:25 v/v). Analyte and internal standard were eluted with 0.2% v/v formic acid in 10 mM ammonium formate and acetonitrile in gradient mode at a flow rate of 0.8 mL/min in 2.0 minute run on Atlantis® dC18 (2.1 X 50 mm, 3 μm) column maintained at 40°C. Quantification was achieved with SCIEX 6500 QTRAP® LC–MS/MS equipped with ESI source in positive mode. MRM pairs m/z 678.4® 147.3 and m/z 455.2→175.1 were used for analyte and internal standard, respectively. Results:Developed method was found to be linear in the range of 0.02-50 ng/mL with regression coefficient greater than 0.998. Accuracy obtained from four different sets of quality control samples analyzed on different days was within acceptable limits of 100± 15% with a precision ≤15%. Analyte and IS recovery was found to be 60%. Pharmacokinetic study performed in wistar rats with 4 µg/rat I.V. dose resulted with maximum concentration (Cmax) of 25.5 ng/mL at 0.08 hr (Tmax) with t1/2 of 2.37 hr.Conclusion:A simple, sensitive, direct LC-MS/MS method was developed and validated using 100µL of serum. Developed method was found to be accurate and precise throughout the calibration range with 5µL injection volume. Solid-phase extraction mechanism has yielded required recovery to achieve 20 pg/mL sensitivity. The method was successfully applied to a rat pharmacokinetic study and which can be applied to human serum samples.