A method of flow injection of the lock mass for accurate mass measurement using electrospray ionization time-of-flight mass spectrometry is described. The reference compound is introduced in the chromatographic effluent via a six-port valve placed post-column, prior to the split connector. Flow injection is performed in such a way that the reference elution peak is superimposed in the total ion current and partially overlaps that of the investigated analyte, allowing independent ionization of the two compounds and thus accurate mass measurement with no ion suppression effects. Different lock mass molecules can be injected in a single analytical run to target various analytes. The performance of this methodology is demonstrated in both isocratic and gradient liquid chromatography modes. The molecular ion of the flow-injected lock mass could also be used as a reference for mass measurement of the in-source fragments of the analytes. Good mass accuracy, within 4 mDa of the theoretical values, was obtained.