Nucleotide-binding proteins play important roles in a variety of biological processes. While ATP- and GTP-binding proteins have been well studied, the systematical identification of UTP-interacting proteins remains under investigated. Here, we developed a chemical proteomic strategy using a biotinylated UTP affinity probe coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) method to enrich, identify and quantify UTP-binding proteins at the entire proteome scale. By performing labeling reactions with low vs high concentrations of UTP probe (10 and 100 μM) or with the UTP probe in the presence of free UTP in stable isotope labeling by amino acids in cell culture (SILAC) experiments, we identified more than 70 potential UTP-binding proteins which are involved in multiple cellular processes, such as translational elongation and protein folding. We also validated the UTP-binding capability of the cytoskeletal protein ACTB by using cellular thermal shift assay (CETSA). Together, we performed a high-throughput chemical proteomics-based analysis to identify, for the first time, UTP-binding proteins in human proteome, which should be applicable for the identification and quantification of UTP-binding proteins in other organisms and tissues.