Most drugs are developed through iterative rounds of chemical synthesis and biochemical testing to optimize the affinity of a particular compound for a protein target of therapeutic interest. This process is challenging because candidate molecules must be selected from a chemical space of more than 10(60) drug-like possibilities(1), and a single reaction used to synthesize each molecule has more than 10(7) plausible permutations of catalysts, ligands, additives and other parameters(2). The merger of a method for high-throughput chemical synthesis with a biochemical assay would facilitate the exploration of this enormous search space and streamline the hunt for new drugs and chemical probes. Miniaturized high-throughput chemical synthesis(3-7) has enabled rapid evaluation of reaction space, but so far the merger of such syntheses with bioassays has been achieved with only low-density reaction arrays, which analyse only a handful of analogues prepared under a single reaction condition(8-13). High-density chemical synthesis approaches that have been coupled to bioassays, including on-bead(14), on-surface(15), on-DNA(16) and mass-encoding technologies(17), greatly reduce material requirements, but they require the covalent linkage of substrates to a potentially reactive support, must be performed under high dilution and must operate in a mixture format. These reaction attributes limit the application of transition-metal catalysts, which are easily poisoned by the many functional groups present in a complex mixture, and of transformations for which the kinetics require a high concentration of reactant. Here we couple high-throughput nanomole-scale synthesis with a label-free affinity-selection mass spectrometry bioassay. Each reaction is performed at a 0.1-molar concentration in a discrete well to enable transition-metal catalysis while consuming less than 0.05 milligrams of substrate per reaction. The affinity-selection mass spectrometry bioassay is then used to rank the affinity of the reaction products to target proteins, removing the need for time-intensive reaction purification. This method enables the primary synthesis and testing steps that are critical to the invention of protein inhibitors to be performed rapidly and with minimal consumption of starting materials.