The use of fluorescence difference decay curves was explored as a way to isolate the decays of components in a complex system. Time-correlated single photon methodology allows one to subtract a “reference” curve from a “sample” curve from a “sample” curve to yield a difference decay curve. To test the feasibility of this method, a model 3-component system (6-carboxyfluorescein, pyranine, and 1-dimethyl-aminoaphtalene-5-sulfonate) was examined. From its complex fluorescence decay curve, the individual decays were obtained by subtracting the appropriate binary solution decay curves. These difference curves coincided with those of the single component systems. Stringent requirements included: use of the same instrumental settings for all solutions, low counting efficiency, avoidance of inner filter effects, absence of energy transfer, and lack of chemical interaction between components. The difference decay method was applied to: 1. Sequential dansylation of serum albumin. Lifetimes of the first two dyes bound are longer than those of the third. 2. Dynamic quenching of quinine fluorescence by chloride ion. When the reference differs from the sample only by having a shorter lifetime, the difference decay curve has a characteristic shape. 3. Quenching of intrinsic protein fluorescence by acrylamide. Bovine serum albumin and liver alchol dehydrogenase were examined. Of the two tryptophans in each protein, one was preferentially quenched and its decay curve was obtained by difference.