This dataset originates from a Proteome Integral Solubility Alteration (PISA) experiment designed to assess how 5-fluorouracil (5-FU) alters protein thermal stability/solubility in MDA-MB-231 cells. Cells were treated with 200 μM 5-FU or DMSO for 2 h, exposed to a temperature gradient, lysed, and soluble proteins from each condition were isolated, digested, and labeled with TMTpro reagents. Labeled peptides were fractionated and analyzed by LC-MS/MS. The raw data were processed with MaxQuant against the human UniProt reference proteome, providing proteome-wide quantitative profiles of 5-FU-induced changes in protein solubility.