A method of mass spectrometry is disclosed that comprises acquiring mass spectral data during a single experimental run or acquisition of a mass spectrometer and determining a first time T during the single experimental run or acquisition for calibrating the mass spectrometer. The step of determining the first time T further comprises determining a time when the mass spectral data being acquired during the single experimental run or acquisition is equal to or below a threshold. The mass spectrometer is then calibrated by introducing calibrant or lockmass ions at the first time T.