Capsaicin, the primary active ingredient and irritant in chili peppers, has been utilized across multiple fields as a food adductive or because of its potential anticancer, antioxidant, anti-inflammatory and metabolic regulatory properties. Despite its diverse uses, the mechanism of action of capsaicin has not been fully revealed. Here, we investigated the changes in the proteome and phosphoproteome of A549 cells upon treatment with capsaicin for different durations and at different doses. Pressure cycling technology (PCT) was applied for rapid sample preparation and digestion, significantly improving the stability of phosphorylated proteins and allowing in-depth phosphoproteome analysis within 6 h with protein inputs of 100 μg. Proteomic and phosphoproteomic alterations can be used to accurately identify perturbations caused by various capsaicin doses and exposure durations. Proteomic analysis revealed that capsaicin administration affected the cell cycle and DNA damage pathways in a time- and dose-dependent manner. Compared with the proteomic changes, more sensitive and rapid alterations were observed in the phosphoproteome, a finding further supported by posttranslational modification (PTM) set enrichment analysis (PTM-SEA) of the phosphoproteomic data. The phosphorylation status of serine protein kinase, Aurora kinase A, and Aurora kinase B changed faster than their protein expression. Overall, the findings here identify the proteomic and phosphoproteomic alterations caused by capsaicin, providing new insights for multiomics analysis to elucidate chemical perturbations.