Deprotonation reactions of peptide and protein ions have been studied by introducing volatile reference bases at atmospheric pressure between an electrosonic spray ionization (ESSI) source and the inlet of a hybrid quadrupole time-of-flight mass spectrometer. This new setup offers the unique possibility to measure the apparent gas-phase basicity GBapp of multiply charged ions by a bracketing approach. A very good agreement has been obtained with reference values obtained by Fourier transform-ion cyclotronic resonance (FT-ICR), validating our approach. The measurements were then extended to larger biomolecules such as insulin and myoglobin in native and denaturing buffers. The main advantages of this methodology are measurements at atmospheric pressure with good sensitivity (for concentrations less than 10 microM in denaturing or nondenaturing buffer), very good precision (less than 2%), and in a short time (less than 30 min to screen up to 23 volatile reference bases).