The quantitative photophysical description of matrix-assisted laser desorption ionization (MALDI) is explored by using a wide range of optical pumping rates established by 22 ps and 4 ns laser pulse lengths. Furthermore, the sample morphology dependence of ionization was tested on pellets compacted at different pressures, yielding surfaces of varying roughness. Mass spectra acquired from 2,5-dihydroxybenzoic acid pellets showed an increase in the matrix ionization threshold fluence with increasing pressure during pellet formation. Sample surfaces produced at higher pressure exhibited lower fractal dimension. This fractal coarsening is suggested to be behind the elevated ion formation threshold in these samples. Compared with conventional nanosecond pulses, the ionization threshold values were consistently higher by a factor of similar to2 for the picosecond laser. The DHB matrix was dramatically more likely to yield fragment ions when irradiated with the longer laser pulse, indicating significant differences in the primary ionization mechanism. Furthermore, fragmentation escalated with the increase in laser fluence for both lasers, although this effect was significantly more pronounced for nanosecond pulses. Dried droplet samples of sinapinic acid and alpha-cyano-4-hydroxycinnamic acid also showed higher fragmentation with the longer laser pulse. Different optical pumping rates and relaxation channels are thought to be responsible for the significant difference in the yield of matrix molecular ions. For small analytes (less than or equal to2000 Da), molecular ion signal was easily acquired with both lasers, whereas for large molecules (greater than or equal to5000 Da), the analyte ion yield was low or even vanishing with the picosecond laser. The nanosecond laser produced ions from the small and large analytes with comparable yields. These observations are compatible with the assumption that the longer laser pulse can sequentially desorb and ionize analyte molecules due to the extended interaction of the laser pulse and the MALDI plume, whereas the shorter pulse can only desorb the analyte molecules but cannot promote their ionization due to its limited temporal overlap with the plume.