The ion pair neutralization model can provide an explanation for the observation that the total numbers of ions detected by the MS detector are roughly constant irrespective of the molar ratio of an analyte over matrix in a MALDI MS sample and the identity of the analyte. Taking the analysis of BTPP-Cl with MALDI MS as an example, when the sample composition was 0.1 pmol analyte in 25 nmol CHCA, the ion counts of BTPP + and [CHCA+H] + were 0.52 × 10 8 and 1.8 × 10 9 , respectively; when the analyte was increased to 3 pmol, the ion counts were 11 × 10 8 and 1.4 × 10 9 , respectively; and when analyte was 30 pmol, the ion counts were 28 × 10 8 and 0.29 × 10 9 , respectively. These data indicate that the total number of ions including those from the analyte and matrix do not change significantly when the ratio of analyte over matrix changes significantly. In addition, it was observed that the total number of ions does not exceed the total number of matrix ions obtained with pure matrix. In certain ranges of analyte concentration in the sample, as the concentration increases, the number of analyte ions increases, but it is at the cost of the number of matrix ions, which decreases. Similar experimental observations were reported for other ionic compounds and peptides. In the context of the ion pair thermal model, three factors may be accountable for the observations. One is that the fraction of molecules including ion pairs and neutral molecules of analyte and matrix that have sufficient thermal energy to render free ion generation is set by the plume temperature. At certain range, the analyte over matrix ratio has little influence on the fraction number. The other is that when the concentration of ion pairs that have sufficient energy to be separable by the ion extraction potential to become free ions dispersed in the plume of mainly neutral matrix molecules exceeds certain limit, they are more likely to collide with each other and become neutral molecules via proton transfer.