Copper is an essential trace element critical to human health. As a redox-active cofactor in metalloproteins, copper catalyzes a broad range of reactions that play vital roles in fundamental biological functions, including respiration, antioxidant defense, bone formation, or iron homeostasis. Increasing evidence suggests that mammalian cells maintain a kinetically labile pool of monovalent copper(I), which is tightly buffered in the subfemtomolar regime. To devise chelators suitable for probing the subfemtomolar activity of cytosolic copper, we created a family of high-affinity Cu(I) ligands based on phosphine-sulfide-stabilized phosphine (PSP) binding motifs. Comprised of molecular architectures with varying degrees of conformational flexibility, the chelators offer tunable Cu(I) dissociation constants from the femto- to subzeptomolar range while effectively discriminating against other biologically relevant trace metals including Zn(II), Fe(II), and Mn(II), even at millimolar concentrations. Moreover, integrating a PSP-binding motif within a donor-acceptor fluorophore platform yielded a Cu(I)-selective emission-ratiometric probe, crisp-17, for visualizing dynamic changes in intracellular copper availability by two-photon excitation microscopy. Taking advantage of the PSP ligand family, we determined the Cu(I) affinity of a metallochaperone and probed the thermodynamic availability of Cu(I) in mammalian cell lysate by size exclusion chromatography (SEC) integrated with ICP-MS for precise elemental quantification. Coupled with 2D-chromatographic separations, we further developed a robust analytical approach for the label-free speciation analysis of cell lysate and other complex biological mixtures. Altogether, these studies paint a unified picture where cells maintain a dynamic yet tightly controlled copper pool that is buffered at low attomolar levels through a polydisperse buffer of bioligands.