Proteins are crucial nutrients for humans, but why are small amounts of proteins excreted through urine? Although healthy individuals have low levels of protein in their urine, the variety of proteins present is diverse. These proteins may represent unnecessary waste products or even have toxic effects on the body, necessitating their excretion. Proteins possess complex structures and perform various functions compared to small-molecule metabolites. Even a minor alteration at the molecular level can impact their subsequent biological functions. By conducting a comprehensive analysis of molecular-level modifications in the proteomes of plasma and urine, differences between the two can provide insights into the presence of proteins in urine. In this chapter, we collected urine and plasma samples from a total of nine healthy individuals. High-resolution tandem mass spectrometry and unlabeled quantitative proteomics techniques were employed to analyze the samples. The nonlimiting modification identification algorithm was used to compare molecular modifications between the two sample types as a whole. The findings revealed a higher incidence of cysteine (Cys) modification to dehydroalanine (Dha) in the urine proteome compared to plasma. The molecular modification [Cys → Dha] disrupts the disulfide bond of the original protein, thereby affecting or even altering the structure and biological function of the protein. Consequently, this study uncovered variations in overall proteome modifications between plasma and urine, highlighting that protein structure may undergo irreversible changes due to differences in protein molecule modifications. As a result, proteins that have lost their functionality or even toxic proteins are excreted from the bloodstream into the urine.