Abstract Protein-protein interactions (PPIs) represent the fundamental components of highly complex and dynamic net- works that govern nearly all cellular processes. The strength of biologically relevant PPIs varies widely from near permanent interactions with sub-nM dissociation constants to weak transient interactions with mM disso- ciation constants. Despite the importance of transient PPIs for many key processes governing cell functions, methods for their in situ characterization are still lacking. To address this problem, we will utilize our modulata- ble fluorescent proteins (FPs) for background-free signal recovery and quantification to develop Modulation Transfer-Förster Resonance Energy Transfer (MT-FRET). Modulating the Donor (a photoswitchable FP, excit- ed at both 488 and 405 nm simultaneously) excitations encodes the sum and difference of the two lasers’ modulation frequencies on donor emission. This modulation transfers to the acceptor, which itself is modulated with 900nm excitation, all in a widefield, epifluorescence geometry. The bound complex, donor, and acceptor signals all appear at unique modulation frequencies with amplitudes that are directly proportional to the con- centration of each. This MT-FRET approach then yields true background-free equilibrium constants, even of weak, transient PPIs, as unbound fluorophore signals appear at different modulation frequencies from that of the complex. Applicable to both freely diffusing and bound species, this is a key advance over existing ap- proaches for determining PPI interactions as it suppresses the overwhelming background characteristic of transient PPIs and those in live cells. Specifically, we will apply these general methods to characterize selected proteins involved in dynamic trafficking of copper in vitro and in live cells. The materials and methods devel- oped will not be limited to the characterization of PPIs relevant to copper homeostasis, but will simultaneously provide a more general set of tools, methods, and overall framework for surveying and measuring transient PPIs within the complex environment of live cells.