Due to a lack of novel therapies and biomarkers, the clinical outcomes of osteosarcoma patients have not significantly improved for decades. The advancement of mass spectrometry (MS), peptide quantification, and downstream pathway analysis enables the investigation of protein profiles across a wide range of input materials, from cell culture to long-term archived clinical specimens. This can provide insight into osteosarcoma biology and identify candidate biomarkers for diagnosis, prognosis, and stratification of chemotherapy response. In this review, we provide an overview of proteomics studies of osteosarcoma, indicate potential biomarkers that might be promising therapeutic targets, and discuss the challenges and opportunities of mass spectrometric-based proteomics in future osteosarcoma research.