Amorphous 3 nm large ultrasmall (TiO) and 7 nm large anatase (TiO) nanoparticles (NPs) were successfully prepared and characterized by TEM, FTIR, DRX, UV-vis, and DLS techniques. The MALDI-TOF/MS was shown to be effective in assessing the surface chemistry but fragmentation processes precluded its use for evaluation of particle size distribution. In fact, the laser causes the fragmentation not only of amorphous TiO NPs but also of the material subjected to heat treatment and crystallization at 450 °C for 4 h upon interaction with the DHB matrix and TFA ionizing agent. No significant difference could be observed in the spectrum by varying the particle size, indicating the high stability of the TiO dimer and its low aggregates in the gaseous phase. In short, MALDI-TOF/MS is effective for the direct analysis of nanoparticle surfaces, especially the interaction of functionalizing molecular species with the inorganic components, which in combination with the other techniques demonstrated to be ideal for the detailed characterization of nanomaterials.