Abstract: The optimum conditions for measuring protein molecular weight were studied using matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).MethodThree different matrices,viz.,sinapic acid (SA),α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) were tested for determination of molecular weight of bovine serum albumin (BSA) and the effects of different sample concentration were observed on quantification of protein samples.Result The results demonstrated that 1 μg protein samples and sinapic acid (SA)matrix dissolved in water solvent (containing 0.1% trifluoroacetic acid and 50% acetonitrile to generate the concentration of 10 mg/mL) produced the best quality of MALDI-TOF-MS spectra.The sample required to determine the exact quantity was optimized as 500 ng.ConclusionThis technique is convenient,rapid,sensitive and reliable for determining protein molecular weight.The optimal combination was determined to obtain optimum mass spectra,necessary for obtaining accurate molecular weight of highly complicated protein samples.