Abstract: The functional state of a cell is largely determined by the spatiotemporal organization of its proteome. Technologies exist for measuring particular aspects of protein turnover and localization, but comprehensive analysis of protein dynamics across different scales is possible only by combining several methods. Here we describe tandem fluorescent protein timers (tFTs), fusions of two single-color fluorescent proteins that mature with different kinetics, which we use to analyze protein turnover and mobility in living cells. We fuse tFTs to proteins in yeast to study the longevity, segregation and inheritance of cellular components and the mobility of proteins between subcellular compartments; to measure protein degradation kinetics without the need for time-course measurements; and to conduct high-throughput screens for regulators of protein turnover. Our experiments reveal the stable nature and asymmetric inheritance of nuclear pore complexes and identify regulators of N-end rule-mediated protein degradation.Systematic monitoring of proteome dynamics would require simultaneous measurement of protein turnover and subcellular trafficking at the single-cell and population scales. The importance of protein turnover was introduced in 1942 by Schonheimer, who noted that "all constituents of living matter, whether functional or structural, of simple or of complex constitution, are in a steady state of rapid flux". Protein homeostasis is now understood as a balance between protein synthesis, through transcription and translation, and protein degradation, through processes such as proteasomal and lysosomal degradation, tuned in response to intrinsic and extrinsic inputs. Alterations in protein turnover are observed in aging organisms and underlie various diseases. Deregulated degradation of cell cycle control proteins such as the p53 tumor suppressor plays a critical role in many forms of human cancers. Abnormal trafficking and degradation of a mutant form of a chloride ion channel causes cystic fibrosis. Moreover, accumulation of specific proteins is linked to neurodegenerative disorders such as Alzheimer's, Parkinson's and Huntington's diseases. Therefore, understanding protein turnover and mobility could provide new strategies for targeted clinical interference to treat such diseases.