Disease-relevant phenotypic screening directly identifies ligands that modulate useful biology and constitutes a promising approach to discovery of novel pharmaceutical treatments. Beyond identifying chemicals with desirable effects, it is important to identify the target(s) and mechanisms that drive the desirable phenotype in complex cellular systems. However, determining the relevant target(s) of phenotypically active ligands has often proven slow or impossible, hampering drug discovery and development. Recently, a few methodologies have emerged that enable detection of target engagement in cells, but most of them require prior chemical modification of either biologically active compounds or proteins. Here, we reported a novel cytosolic proteome & affinity-based target identification platform (CPATI), which is an unbiased, label-free, and modification-free approach. We applied CPATI to identify the candidate target protein(s) of three compounds with Jurkat cells. First, native size-exclusion chromatography (SEC) was used to separate cellular cytosol isolated from Jurkat cell lysis. Second, using affinity-selection technology with our two-dimensional LC-MS system, three compounds were screened with 170 SEC fractions. Cytosolic fractions identified to have specific ligand binding were analyzed via quantitative proteomics. A combination of the protein-bound ligand recovery and target protein SEC elution profile revealed potential targets of test compounds. Third, thermal shift experiment was conducted to identify proteins with elevated Tm in the presence of ligands, yielding a short list of target proteins. Fourth, the target proteins were selected for recombinant protein production and were validated in a binding assay with ligands. Finally, top target proteins were recommended for further validation. Three control ligands, compound A, compound B, and compound C were identified to bind specifically with Jurkat cytosolic fractions. Their associated target proteins NUDT1 (compound A), HSP90 (compound B), and PAK4 (compound C) were identified from the Jurkat cytosol as their top candidate targets, respectively. compound A and compound B had similar binding affinity (Kds) with specific cytosolic fractions and recombinant proteins NUDT1 and HSP90AA1/AB1, respectively. PPP3A-HSP90-CCT complexes were also identified. Compound C is an ATP competitive kinase inhibitor and was identified to associate with seventeen target proteins. Three recombinant proteins PRKACB, PRKCQ, and STK38 were confirmed as compound C-bound target proteins. We also compared thermal shift experiments using compound C with compound C-bound cytosolic fractions and jurkat crude cytosol. Former samples demonstrated obviously advantage over later samples with short potential target proteins and less false positive. This technology potentially provides a broad application in target and biomarker identification from cells and tissues.