Thermal proteome profiling (TPP) enables unbiased identification of protein drug targets and potential off-target effects. The technique relies on monitoring the ligand-induced thermal stability changes across the proteome in a protein’s native state. Cellular Thermal Shift Assay (CETSA)1 and Proteome Integral Solubility Alteration (PISA)2 are two leading MS-based target engagement studies utilising the TPP principle. CETSA-MS measures the shift in the protein's melting curve, based on ligand-induced solubility changes. In a similar approach, PISA-MS measures the difference in the integral protein abundances, without the curve fitting required in CETSA. PISA overcomes the ineffectiveness of CETSA in characterising proteins whose thermal profile does not fit into a sigmoidal melting curve. To overcome the cost of isobaric labels, a requirement of a high-resolution instrument to deconvolute isobaric masses, and the need for complex data analysis for curve fitting during CETSA, we adopted a label-free approach to analyse PISA pools by data-independent acquisition (PISA-DIA). The lysate PISA-DIA analysis workflow was tested on four overlapping temperature gradients containing eight temperatures each (37 °C – 49 °C, 44 °C – 56 °C, 51 °C – 63 °C, and 58 °C – 70 °C ). This is the first PISA-MS study of its kind known to use overlapping gradients as a means of increasing the resolution by providing additional data points to establish the precise drug target under investigation. For this purpose, human multiple myeloma cells expressing BCL-xL (BCL2L1) were lysed and either treated with an established BCL-xL inhibitor (Venetoclax, 1 µM and 2 µM) or DMSO in quadruplicates. PISA pools were analysed by diaPASEF on a timsTOF Pro instrument (30 min gradient/sample) following USP3 digestion3 and StageTip peptide cleanup. The PISA-DIA analysis identified the known drug target (BCL-xL) in both drug concentrations within the 44 °C – 56 °C gradient. This benchmarking study confirms the robustness, suitability and cost-effectiveness of the current PISA-DIA workflow for routine target engagement studies.