Determining protein targets directly bound by drug molecules remains challenging. Here we present the isothermal shift assay, iTSA, for rapid unbiased identification of drug targets. Compared with thermal proteome profiling, the prevailing method for target engagement, iTSA offers a simplified workflow, 4- fold higher throughput, and a multiplexed experimental design with higher replication. We demonstrate its application to determination of target engagement for several kinase inhibitors in lysates and living cells.