Cysteine oxidation is a central mechanism of redox signalling. However, the comprehensive characterization of these modifications remains limited by the sensitivity and proteome coverage of current mass spectrometry-based redox proteomics workflows. To address these challenges, we developed “Two-Step-One-Pot” cysteine enrichment strategy based on sequential isotopic labelling of reduced and oxidized cysteine residues using thiol-specific click chemistry probes. This approach enables differential cysteine labelling within a single sample, eliminating the need of separating light and heavy tagged replicates and requiring only minimal starting sample amounts. Concomitantly, systematic optimization of labelling and enrichment resulted in improved identification of cysteine-containing peptides. Application of the workflow to redox proteome profiling of cells showed increased sensitivity and improved protein sequence coverage compared to our previously published approaches. In summary, we present a novel platform for quantitative redox proteomics which enables in-depth profiling of cysteine-mediated redox regulation.