Reproducible, comprehensive phosphopeptide enrichment is essential for studying phosphorylation-regulated processes. Here, we describe the application of hyper-porous magnetic TiO2 and Ti-IMAC microspheres for uniform automated phosphopeptide enrichment. Combining magnetic microspheres with a magnetic particle-handling robot enables rapid (45 min), reproducible (r2 ≥ 0.80) and highfidelity (>90% purity) phosphopeptide purification in a 96-well format. Automated phosphopeptide enrichment demonstrates reproducible synthetic phosphopeptide recovery across 2 orders of magnitude, “well-to-well” quantitative reproducibility indistinguishable to internal SILAC standards, and robust “plate-to-plate” reproducibility across 5 days of independent enrichments. As a result, automated phosphopeptide enrichment enables statistical analysis of label-free phosphoproteomic samples in a highthroughput manner. This technique uses commercially available, off-the-shelf components and can be easily adopted by any laboratory interested in phosphoproteomic analysis. We provide a free downloadable automated phosphopeptide enrichment program to facilitate uniform interlaboratory collaboration and exchange of phosphoproteomic data sets. P protein phosphorylation is an important medium for cellular signal transduction. Protein kinases and phosphatases are often deregulated in disease, and pharmacological modulation of phosphorylation-dependent signal transduction is an active area of research. Consequently, quantitative analysis of pathological phosphoproteomes is of substantial interest to the biological research community. Liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) is a powerful technology used to characterize and quantify phosphorylated proteins. However, given the low stoichiometric abundance of phosphorylated residues within the proteome, phosphopeptide enrichment is required for comprehensive phosphoproteomic analysis. Due to the complexity and dynamic range of the phosphoproteome, sample prefractionation is commonly used to obtain comprehensive coverage. Popular prefractionation techniques include strong cation exchange (SCX), hydrophilic interaction liquid chromatography (HILIC), and electrostatic repulsion hydrophilic interaction chromatography (ERLIC). Subsequent affinity-based phosphopeptide enrichment commonly employs metal dioxides (such as titanium and zirconium) or immobilized metal ion affinity chromatography (IMAC). Despite the established performance of these matrices, a limited number of workflows have been developed for automation. Consequently, existing affinity enrichment methodologies still operate in either manual batch-mode or as manual prepacked spin-columns. Extensive upstream chromatographic separation massively expands the number of samples for phosphopeptide enrichment processing. Successful approaches to avoid prefractionation have been reported for samples with limited input variables. However, these methods do not facilitate the uniform parallel phosphopeptide enrichments required for the increased numbers of biological variables/replicates now common in phosphoproteomics. Such multivariate expansions are further exacerbated by the contemporary trend toward multisite proteomic projects. These endeavors require interlaboratory integration of large sample sets and demand invariable sample processing across disparate users. Collectively, these factors increase both the number of simultaneous phosphopeptide enrichments required for phosphoproteomic analysis and the demand for unified sample-to-sample enrich-