A method for calibrating a time-of-flight mass spectrometer is disclosed. The method includes determining the time-of-flight values, or values derived from the time-of-flight values for a calibration substance at each of a plurality of different addressable locations on a sample substrate. Then, one of the addressable locations on the substrate is identified as a reference addressable location. A plurality correction factors are then calculated for the respective addressable locations on the substrate using the time-of-flight value, or a value derived from the time-of-flight value.