To provide an objective measure of the correlation between the internal energy content of ions generated by matrix-assisted laser desorption ionization (MALDI) and the matrix properties, a series of well-characterized benzyl-substituted benzylpyridinium salts were used as thermometer molecules (TMs). To determine the internal energy variations of analyte ions, the survival yields of TM molecular ions were measured in three different matrixes, alpha-cyano-4-hydroxycinnamic