The spatial changes of lipids and lipid-related proteins play a vital role in the reflection of osteoarthritis. Due to sample preparation difficulties, the hallmark of spatial changes in lipids and proteins remains less well known. The aim of this study is to use advanced mass spectrometry imaging techniques to discover the proteins, and lipids and their functionality change at a spatial level. We collected osteochondral tissue samples stratified according to disease severity, from 10 knee OA patients who underwent knee replacement surgery. We defined the lipid and related protein changes using the spatial multi-omics technique, including spatial proteomics, spatial lipidomics and functional mass spectrometry imaging technique. We confirmed our results using immunohistochemistry and laser-microdissection guided lipidomics and proteomics. Our results find a characteristic distribution of lipids and lipid-related proteins in the osteochondral unit. We also demonstrated that the functionality of phospholipase A2 was reactivated on the surface of the cartilage surface. In conclusion, our results suggest that the multiple lipids and lipid-related protein changes can reflect the disease progression.