Updated project metadata. Manipulating protein synthesis is commonly applied in bio-related research to uncover protein functions and cellular activities. Multiple inhibitors with distinct mechanisms are widely used, but it is extraordinarily challenging to systematically determine and evaluate their inhibition efficiencies, especially for individual proteins. Newly synthesized proteins are direct products of protein synthesis, and thus their comprehensive and quantitative analysis provides a unique opportunity to study protein synthesis inhibition. Here, we systematically investigate and evaluate popular inhibitors through global quantification of nascent proteins in several types of human cells. The same inhibitor results in dramatic differences in the inhibition efficiencies for different proteins, and each inhibitor exhibits unique synthesis inhibition preference. Overall, nucleolar and ribosomal proteins have relatively higher inhibition efficiencies, while the efficiencies of proteins in the peroxisome and the Golgi are lower. The sensitivity of proteins to the inhibition can reflect the characteristics of each cell type. Moreover, proteins generally resistant to the inhibition have lower translation efficiency, among which chaperones are overrepresented. Systematic evaluation of protein synthesis inhibition aids in better understanding protein synthesis regulation and provides valuable information to the biology and biomedicine communities.