Loss-of-function approaches such as gene knockout/knockdown have been widely used to identify the proteins that are necessary to organize various cellular behaviors. However common approaches, such as genetics or RNAi, change the cellular environment permanently. Over days and weeks compensatory changes can accumulate, alter the cell's physiology, and confound conclusions. Current approaches that act on faster (seconds) timescales have limitations in their specificity, are often irreversible, hrd to titrate, or difficult to apply to a wide range of target proteins. Moreover, in cases where most components of a pathway have been identified, questions shift towards timing "At what stage is my protein involved?" and level "Is cell behavior sensitive to the concentration of my protein?" Answers to such questions require the ability to trigger, set, reverse, and monitor the extent of protein inactivation with high precision - an area where traditional methods fail, and new tools are required. Towards this end, we are developing an optogenetics based loss-of-function method termed DeLIGHT (Depletion with LIGHT), as a much-needed tool for the growing field of single-cell biochemistry. We have recently made breakthroughs in developing a light-induced protein interaction module and will here combine this tool with an 'anchor away' strategy to reversibly sequester proteins at inert intracellular locations. Our technique is generalizable, acute, and reversible, enabling significantly greater control over protein activity than existing inactivation methods. DeLight makes it possible to measure single-cell responses at user-defined intermediate concentrations of a protein of interest, where the extent of inactivation can be visually monitored and set by the user in an interactive fashion. We propose to establish and optimize this approach in 3 model systems - Dictyostelium, budding yeast, and mammalian cells to test the modularity of this system, demonstrate its versatility, and provide tools for immediate application by the cell biology community. Traditional techniques for inactivating gene function have proven to be powerful at identifying proteins involved in a biological process, but these approaches lack generality, reversibility, and titratability. Here we develop a light-based perturbative technique that addresses all of these needs. This powerful approach could enable the biomedical research community to better dissect the cell signaling processes that underlie human diseases.