Multivalent interactions are common in biological systems and are also widely deployed for targeting applications in biomedicine. A unique feature of multivalent binding is "superselectivity". Superselectivity refers to the sharp discrimination of surfaces (e.g., on cells or cell compartments) by their comparative surface densities of a given receptor. This feature is different from the conventional "type" selectivity, which discriminates surfaces by their distinct receptor types. In a broader definition, a probe is superselective if it converts a gradual change in any one interaction parameter into a sharp on/off dependency in probe binding.This Account describes our systematic experimental and theoretical efforts over the past decade to analyze the determinants of superselective binding. It aims to offer chemical biologists, biophysicists, biologists, and biomedical scientists a set of guidelines for the interpretation of multivalent binding data, and design rules for tuning superselective targeting. We first provide a basic introduction that identifies multiple low-affinity interactions and combinatorial entropy as the minimal set of conditions required for superselective recognition. We then introduce the main experimental and theoretical tools and analyze how salient features of the multivalent probes (i.e., their concentration, size, ligand valency, and scaffold type), of the surface receptors (i.e., their affinity for ligands, surface density, and mobility), and of competitors and cofactors (i.e., their concentration and affinity for the ligands and/or receptors) influence the sharpness and the position of the threshold for superselective recognition.Emerging from this work are a set of relatively simple yet quantitative data analysis guidelines and superselectivity design rules that apply to a broad range of probe types and interaction systems. The key finding is the scaling variable xS which faithfully predicts the influence of the surface receptor density, probe ligand valency, receptor-ligand affinity, and competitor/cofactor concentrations and affinities on superselective recognition. The scaling variable is a simple yet versatile tool to quantitatively tune the on/off threshold of superselective probes. We exemplify its application by reviewing and reinterpreting literature data for selected biological and biomedical interaction systems where superselectivity clearly is important.Our guidelines can be deployed to generate a new mechanistic understanding of multivalent recognition events inside and outside cells and the downstream physiological/pathological implications. Moreover, the design rules can be harnessed to develop novel superselective probes for analytical purposes in the life sciences and for diagnostic/therapeutic intervention in biomedicine.