In surface-assisted laser desorption/ionization mass spectrometry using titania (TiO₂) films (TiO₂-SALDI-MS), one of the most important aspects is the absence of matrix interferences in the low-mass region. Therefore, in TiO₂-SALDI, the detectable mass range for small biomolecules, pharmaceutical compounds, amino acids, and oligopeptides can be extended to below m/z 500. The physicochemical properties of titania are dependent on the crystalline form of the material; however, thus far, very little attention has been given to the effect of the crystalline form of titania on titania-based SALDI-MS. We investigated the influence of crystalline forms on the desorption/ionization efficiency of TiO₂-based SALDI-MS using different crystalline forms such as rutile, anatase, and a non-crystalline amorphous structure. On the basis of survival yield measurements using benzylpyridium chloride, and the desorption/ionization efficiency of peptides (angiotensin II and gly-gly-tyr-arg) and saccharides (cellobiose and hexa-N-acetyl-chitohexaose), it was found that the anatase-type TiO₂ is suitable for the TiO₂-based SALDI-MS. Rutile-type TiO₂ lost the SALDI activity compared with TiO₂ in other crystalline forms, although rutile-type TiO₂ showed the highest UV absorbance.