During and after surgical resection, it is imperative to demonstrate complete removal of tumor as remaining tumor cells will likely lead to recurrence. In fact, it is common that cancerous or precancerous cells are “left behind” because they are not recognized under normal light microscopy. Here, we show how imaging mass spectrometry detects cancer-related proteins beyond the “histologically normal” tissue. We are now proving that such proteins represent the “molecular margin” using traditional molecular biological experimentation.