Using polyethylene glycol (PEG) as internal reference, electrospray ionization (ESI)/time-of-flight mass spectrometry (TOF-MS) was developed to determine the accurate mass of the protonated molecular ions of five erythromycin analogue antibiotics. Compared with the theoretical values, all relative errors were less than 5×10^-^6. PEG can form three adduct ions with K^+, Na^+, H^+, respectively, and may appear with single kind of adduct ions in suitable experiment conditions. PEG was recommended as a general internal reference compound. Besides, two-peak calibration is preferred to the multi- peak calibration. Scan mass width and scan speed were also studied, 104～144amu 5 seconds was recommedned. Using polyethylene glycol(PEG) as internal reference, ESI/TOF mass spectrometry can determine the accurate mass of the protonated molecular ions of the unsteady basic compounds. This method is simple and expeditious.

Key words: TIME-OFF FLIGHT MASS SPECTROMETRY, ERYTHROMYCINUM, ANTIBIOTICS