Titanium dioxide, titania, is used as an affinity support for enriching phosphopeptides in mass spectrometry-based phosphoproteomics. In this study, a variety of titania particles were prepared by calcination at different temperatures or were obtained from different manufacturers, and relationships between their physico-chemical properties and the selectivity in phosphopeptide enrichment was examined. Tryptic peptides from three phosphoproteins were employed for phosphopeptide enrichment by aliphatic hydroxyl acid-modified metal oxide chromatography (HAMMOC) using titania in the presence of lactic acid. The recovery of phosphopeptides increased with increasing calcination temperature, up to 500°C, while the number of identified non-phosphopeptides decreased. For titania calcined at 800°C, the retention of phoshopeptides as well as non-phosphopeptides was significantly reduced in the presence of lactic acid. Various physico-chemical parameters, such as crystalline form, pore size and surface area were examined for these calcined titania beads, as well as commercial titania beads. The findings indicate that the weight-loss from 130°C to 800°C, as determined by thermal analysis, was well correlated with the selectivity of phosphopeptides by HAMMOC. Based on these results, optimized titania beads were prepared and applied to 100 μg of a HeLa cell extract. More than 1,000 phosphorylation sites were successfully identified with a false positive rate of 1.4%, whereas 455 phosphorylation sites were identified with a false positive rate of 4.1% when commercially available titania beads were used for HAMMOC.