Although positive thermometer ions are widely used for evaluating the internal energy distribution of gas-phase ions, negative thermometer ions have not yet been proposed. In this study, phenyl sulfate derivatives were tested as thermometer ions to characterize the internal energy distribution of ions produced by electrospray ionization (ESI) in the negative mode because the activation of phenyl sulfate preferentially undergoes SO3 loss, providing a phenolate anion. The dissociation threshold energies for the phenyl sulfate derivatives were determined using quantum chemistry calculations at the CCSD(T)/6-311++G(2df,p)//M06-2X-D3/6-311++G(d,p) level of theory. The values for the appearance energies of the fragment ions of the phenyl sulfate derivatives depend on the dissociation time scale in the experiment; therefore, the dissociation rate constants of the corresponding ions were estimated using the Rice-Ramsperger-Kassel-Marcus theory. The phenyl sulfate derivatives were used as thermometer ions to determine the internal energy distribution of negative ions activated by the in-source collision-induced dissociation (CID) and higher-energy collisional dissociation. Both mean and full width at half-maximum values increased with increasing ion collision energy. In the in-source CID experiments, the internal energy distributions obtained by phenyl sulfate derivatives are similar to that when all voltages are mirrored, and the traditional benzylpyridinium thermometer ions are used. The reported method will aid in determining the optimum voltage for ESI mass spectrometry and the subsequent tandem mass spectrometry of acidic analyte molecules.