Cells employ a variety of strategies to maintain proteome homeostasis. Beginning during protein biogenesis, the translation machinery and a number of molecular chaperones promote correct de novo folding of nascent proteins even before synthesis is complete. Another set of molecular chaperones helps to maintain proteins in their functional, native state. Polypeptides that are no longer needed or pose a threat to the cell, such as misfolded proteins and aggregates, are removed in an efficient and timely fashion by ATP-dependent proteases. In this review, we describe how applications of single-molecule manipulation methods, in particular optical tweezers, are shedding new light on the molecular mechanisms of quality control during the life cycles of proteins. Copyright © 2014 by Annual Reviews. All rights reserved.