Technologies that enable intraoperative tissue analysis and diagnosis are critically needed to guide surgical procedures and improve patient outcomes. Here we demonstrate the MasSpec Pen technology as a handheld device, integrated with a mass spectrometer, for rapid and direct molecular analysis of biological samples. Coupled with statistical modeling, we show that this rapid (<15s), non-destructive technology enables tissue classification and cancer diagnosis with high accuracy. Today’s presentation will describe recent developments and results translating MasSpec Pen technology integrated with a Thermo Scientific™ Orbitrap™ mass spectrometer to the clinic and operating rooms for in vivo tissue analysis and diagnosis. Technical developments and diagnostic performance in cancer surgery will also be discussed, as well as new opportunities and applications in the clinical setting.