A well-adapted model system to study aging of postmitotic mammalian cells is the analysis of the chronological life span of Saccharomyes cerevisiae, where the length of time that a population of non-dividing remains viable under stationary growth conditions is being monitored. In support of the free radical theory of aging, it has been shown that during chronological yeast aging, reactive oxygen species (ROS) accumulate and cause damage to cellular macromolecules. However, neither the precise onset of oxidative damage nor the type(s) of ROS have been identified. Yet this knowledge is crucial to determine whether oxidative damage is cause or consequence of aging. Here we applied our highly sensitive global protein thiol trapping technique termed OxICAT to monitor and precisely quantify the extent of oxidative thiol modifications during chronological yeast aging. We found that during exponential growth, the vast majority of thiol-containing yeast proteins are reduced. The proteins maintain their reduced thiol status even upon challenge with high concentrations of physiological oxidants such as H2O2, superoxide and NO·, indicating that effective antioxidant systems exist that quickly restore the cellular redox balance. Most of the yeast proteins remain reduced for the first two days in stationary growth. Then, however, they rapidly accumulate in their thiol-oxidized state. At day 4 in stationary growth, most proteins with redox sensitive cysteines were fully oxidized, although more than 70% of yeast cells were still alive. This result strongly suggests that older yeast cells lose their capacity to maintain their cellular redox balance. To evaluate whether the onset of this redox collapse correlates with the life span of yeast cells, we cultivated our yeast strain in glucose-restricted medium, a caloric restriction (CR) treatment that significantly extends yeast life span. Under these conditions, we found that the onset of protein oxidation was significantly delayed suggesting that maintaining the cellular redox balance might indeed contribute to the life span of yeast. Clustering analysis of the affected proteins revealed only a few select proteins, whose thiol-oxidation substantially preceded the oxidation of all other proteins. One of these proteins was thioredoxin reductase (TrxR), a highly conserved key component of the cellular redox-balancing machinery, whose reduced redox status is essential for maintaining the redox status of other cellular proteins. This result agreed well with our conclusion that a collapse of the cellular redox balance causes the massive oxidation of redox-sensitive proteins. It remains now to be determined whether the premature oxidation of thioredoxin reductase is a programmed or ROS-induced process and whether it might represent one of the key events that trigger chronological aging in yeast cells.