A method of quantifying a target analyte by mass spectrometry, the method comprising: preparing a single sample by combining a first known quantity of a first calibrator, a second known quantity of a second calibrator, and a third known quantity of a third calibrator in a single sample comprising the target analyte, wherein the target analyte is a steroid, wherein the first calibrator, the second calibrator, and the third calibrator are each different stable isotope analogs of the target analyte, and the target analyte is unlabeled; wherein a quantity range defined by the first, second and third calibrators for the target analyte spans an expected analytical range of the target analyte in the sample; generating a mass spectrometer signal from the single sample using a mass spectrometer comprising a first calibrator signal, comprising a second calibrator signal, comprising a third calibrator signal, and comprising a target analyte signal; wherein the first known quantity, the second known quantity, and the third known quantity are different, and wherein the first calibrator, the second calibrator, the third calibrator and the target analyte are each distinguishable in the single sample by mass spectrometry due to differences in their mass, fragmentation pattern and/or combinations thereof; and quantifying the target analyte in the single sample using the first calibrator signal, the second calibrator signal, the third calibrator signal, and the target analyte signal by obtaining a calibration curve from the first calibrator signal, the second calibrator signal, and the third calibrator signal and quantifying the target analyte using the calibration curve and the target analyte signal. preparing a single sample by combining a first known quantity of a first calibrator, a second known quantity of a second calibrator, and a third known quantity of a third calibrator in a single sample comprising the target analyte, wherein the target analyte is a steroid, wherein the first calibrator, the second calibrator, and the third calibrator are each different stable isotope analogs of the target analyte, and the target analyte is unlabeled; wherein a quantity range defined by the first, second and third calibrators for the target analyte spans an expected analytical range of the target analyte in the sample; generating a mass spectrometer signal from the single sample using a mass spectrometer comprising a first calibrator signal, comprising a second calibrator signal, comprising a third calibrator signal, and comprising a target analyte signal; wherein the first known quantity, the second known quantity, and the third known quantity are different, and wherein the first calibrator, the second calibrator, the third calibrator and the target analyte are each distinguishable in the single sample by mass spectrometry due to differences in their mass, fragmentation pattern and/or combinations thereof; and quantifying the target analyte in the single sample using the first calibrator signal, the second calibrator signal, the third calibrator signal, and the target analyte signal by obtaining a calibration curve from the first calibrator signal, the second calibrator signal, and the third calibrator signal and quantifying the target analyte using the calibration curve and the target analyte signal.