Biomarker is the measurable change associated with a physiological or pathophysiological process, its nature is change. Contrast to the blood which is under homeostatic controls, urine reflects changes in the body earlier and more sensitive therefore is a better biomarker source. Lysine acetylation is an abundant and highly regulated post-translational modification. It plays a pivotal role in modulating diverse biological processes and is associated with various important diseases. Enrichment or visualization of proteins with specific post-translational modifications provides a method for sampling the urinary proteome and reducing sample complexity. In this study, we used anti-acetyllysine antibody-based immunoaffinity enrichment combined with high-resolution mass spectrometry to profile lysine-acetylated proteins in normal human urine. A total of 654 acetylation sites on 335 proteins were identified, including some very low-abundance proteins. This is the first proteome-wide characterization of lysine acetylation proteins in normal human urine. Lysine acetylated proteins in the urine may reflect protein acetylation status in cells. Discarding acetylated proteins may provide a way to modulate levels of lysine acetylated proteins in cells. Our dataset provides a useful resource for the further discovery of the lysine acetylated proteins as biomarker in urine.