The objective of the current studywas to develop and verify LCMSmethod for the estimation of lactose in swab and air sample. The stationary phase was a Supelcosil, 3µm (50 x 4.6 mm i.d., 3ï) column. Themobile phase was prepared bymixing acetonitrile: 5mMammoniumformate (80:20, v/v). Detectionwasmade atm/z 360.3 / 163.2 using ESI Positive ion spray ionization mode. The method was found to be linear in the concentration range of 3.089-51.490 ng/ml. Themethod was successfully applied for estimation of the lactose in swab and air sample. The concentration of sampleswas found to be below detection limit. A typical LCMS analysis is done in less time, resulting in a savings of more than a full day over HPLC methods. In addition to throughput rates, HPLC requires additional time for column equilibration and mobile phase preparation. Another significant factor in HPLC analysis is the handling and costs associated with the purchase and disposal of eluting solvents. LCMS eliminates all of these elements, saving both time and cost.