Automation of chemical synthesis and high-throughput (HT) screening are important for speeding up drug discovery. Here, we describe an automated HT picomole scale synthesis system which uses desorption electrospray ionization (DESI) to create microdroplets of reaction mixtures at individual positions from a two-dimensional reactant array and transfer them to a corresponding position in an array of collected reaction products. On-the-fly chemical transformations are facilitated by the reaction acceleration phenomenon in microdroplets and high reaction conversions are achieved during the milliseconds droplet flight time from the reactant to the product array. Successful functionalization of bioactive molecules is demonstrated through the generation of 172 analogs (64% success rate) using multiple reaction types. Synthesis throughput is ∼45 seconds/reaction including droplet formation, reaction, and collection steps, all of which occur in an integrated fashion, generating product amounts sufficient for subsequent bioactivity screening (low ng to low μg). Quantitative performance was validated using LC/MS. This system bridges the demonstrated capabilities of HT-DESI for reaction screening and label-free bioassays, allowing consolidation of the key early drug discovery steps around a single synthetic-analytical technology.