Proteome Integral Solubility Alteration (PISA) assay is widely used for identifying drug targets, but it is labour-, sample and time-consuming. Aiming at enabling automation and greatly reducing the sample amount, we developed One-Pot Time-Induced (OPTI)-PISA. Here we demonstrate OPTI-PISA performance on identifying targets of multiple drugs in cell lysate and scaling down the sample amount to sub-microgram levels, making PISA method suitable to NanoProteomics.