In this study, we integrate rapid evaporative ionization mass spectrometry (REIMS) with the Harmonic scalpel, an advanced laparoscopic surgical instrument that utilizes ultrasound energy to dissect and coagulate tissues. It provides unparalleled manipulation capability to surgeons and has superseded traditional electrosurgical tools particularly in abdominal surgery, but is yet to be validated with REIMS. The REIMS platform coupled with the Harmonic device was shown to produce tissue-specific lipid profiles through the analysis of porcine tissues in both negative and positive ionization modes. Comparison with other methods of electrosurgical dissection, such as monopolar electrosurgery and CO2 laser, showed spectral differences in the profile dependent on the energy device used. The Harmonic device demonstrated major spectral differences in the phospholipid region of m/z 600–1000 compared with the monopolar electrosurgical and CO2 laser-generated spectra. Within the Harmonic REIMS spectra, high intensities of diglycerides and triglycerides were observed. In contrast, monopolar electrosurgical and laser spectra demonstrated high abundances of glycerophospholipids. The Harmonic scalpel was able to differentiate between the liver, muscle, colon, and small intestine, demonstrating 100% diagnostic accuracy. The validation of the Harmonic device–mass spectrometry combination will allow the platform to be used safely and robustly for real-time in vivo surgical tissue identification in a variety of clinical applications.