The present invention is based on the discovery that drug resistance in microorganisms can be rapidly and accurately determined using mass spectrometry. A mass spectrum of an intact microorganism or one or more isolated biomarkers from the microorganism grown in drug containing, isotopically-labeled media is compared with a mass spectrum of the intact microorganism or one or more isolated biomarkers from the microorganism grown in non-labeled media without the drug present. Drug resistance is determined by predicting and detecting a characteristic mass shift of one or more biomarkers using algorithms. The characteristic mass shift is indicative that the microorganism is growing in the presence of the drug and incorporating the isotopic label into the one or more biomarkers, resulting in change in mass.