Cisplatin and oxaliplatin are widely used anti-tumour chemotherapeutic agents with different spectra of activity. The therapeutic efficacy of such platinum-based drug is believed to, at least in part, result from formation of Pt-DNA adducts, followed by DNA damage response and ultimately apoptosis. However, it remains unclear whether these DNA lesions caused by cisplatin and oxaliplatin elicit distinct reactions in cellular signaling pathways. Here, a label-free comparative proteomic study was performed to profile the protein phosphorylation patterns using Pt-DNA probes with different ligand identities and geometries. Phosphorylated proteins recognizing different cisplatin- and oxaliplatin-DNA lesions were enriched and analyzed on LC-MS/MS. Proteomic analysis revealed that cisplatin mainly affected proteins involved in mRNA processing, while chromatin organization and rRNA processing are two major biological processes influenced by oxaliplatin. Changes to site-specific phosphorylation levels of two proteins YBX1 and UBF1 were also validated by Western blotting. In particular, platinum drug treatment in colon and liver cancer cell lines down-regulated S484 phosphorylation of UBF1, which is an essential transcription factor responsible for ribosomal DNA transcription activation, implying that inhibition of ribosome biogenesis might be involved in the cytotoxic mechanism of platinum drugs. Collectively, these results directly reflected distinct protein phosphorylation patterns triggered by cisplatin and oxaliplatin, and could also provide valuable resources for future mechanistic studies of platinum-based anti-tumour agents.