Abstract: It is generally accepted that extractive electrospray ionization (EESI) source is softer than electrospray ionization (ESI) source, but how soft between them is still unclear. There are some differences existed in the internal energy of these two soft ionization sources. In this study, a method for the determination of the internal energy difference of singly charged cations generated by EESI and ESI was proposed and established. Taking rhodamine B as an example, extractive electrospray ionization mass spectrometry (EESI-MS) and electrospray ionization mass spectrometry (ESI-MS) were used to study the fragmentation behavior of rhodamine B, and the effects of different collision energy on the cracking behavior were investigated. Based on the intensities of the fragment ions at different collision energies generated from ESI-MS/MS and EESI-MS/MS, the formula involving the determination of internal energy difference of these two sources was derived. As a result, the difference of internal energy of singly charged cations generated between these two ion sources was calculated to be 11.5 eV. This study suggested that EESI is a softer source, which can better maintain the native conformation of protein and easier generate cluster ions than ESI. This method not only can provide a reference for the comparison of the internal energy differences of other ion sources, but also provide a reference for the deep understanding of the mass spectra of ions produced by different ion sources. Other types of fragmentation process or multiple charged cations will be further investigated to verify the reliability and veracity of this method.