Measurement of protein phosphorylation plays an essential role in delineating cell signaling pathways. Although the detection of a specific phosphoprotein has been largely accomplished by immunological methods, a specific and sensitive assay to measure total protein phosphorylation level in complex samples such as whole cell extracts has yet to be established. Here, we present a sensitive phosphorylation assay on a microwell plate to determine total protein phosphorylation level calibrated to a phosphoprotein standard. The core of the assay is a reagent termed pIMAGO that is multi-functionalized with titanium ions for its superior selectivity towards phosphorylated proteins and with fluorophores for quantification. The specificity, sensitivity, and quantitative nature of the assay were demonstrated with standard proteins and whole cell lysates. The method was then employed to measure the overall protein phosphorylation level of human cells under different treatments. At last, we investigated the practicability of the assay to serve as a sensitive tool to estimate the amount of phosphorylated samples prior to a mass spectrometry-based phosphoproteomic analysis.