In surgical oncology, decisions regarding the amount of tissue to be removed can have important consequences: the decision between preserving sufficient healthy tissue and eliminating all tumor cells is one to be made intraoperatively. This review discusses the latest technical innovations for a more accurate tumor margin localization based on mass spectrometry. Highlighting the latest mass spectrometric inventions, real-time diagnosis seems to be within reach; focusing on the intelligent knife, desorption electrospray ionization, picosecond infrared laser and MasSpec pen, the current technical status is evaluated critically concerning its scientific and medical practice.