Existing thermal shift-based mass spectrometry approaches, such as TPP and PISA, focus mainly on soluble fractions, often neglecting the insoluble pellet, limiting comprehensive drug–target profiling. In this work, we developed DrPISA (deep eutectic solvent-assisted reverse PISA), a novel strategy that enables direct analysis of protein precipitates by integrating optimized deep eutectic solvents (DESs) into the PISA framework. Combined with LC-MS/MS, DrPISA demonstrated broad applicability across multiple model compounds, including methotrexate (MTX), cyclosporin A (CsA), geldanamycin (GA), panobinostat (PAN), and staurosporine (STS), as well as a natural product celastrol (CEL), highlighting its potential in target identification for bioactive natural products.