Background: Femtosecond (fs) Laser Ionisation Mass Spectrometry (fs-LIMS) on colon tissues are described and investigated using ionization/fragmentation processes in details to present a new application in this study. Linear Time of Flight (L-TOF) mass analyzer was utilized to investigate paraffin-embedded human tissue in this study. The effect of fs laser intensity on the spectral characteristics was investigated and interpreted due to mass spectra obtained using 800 nm wavelength with 90 fs pulses at 1 kHz repetition rate. Objectives: Mass spectra of tissues were recorded from L-TOF system and then analyzed by performing a statistical approach called Principal Component Analysis (PCA). The fs-LIMS method applied is proposed as a new and pioneering technology to analyze tissues using L-TOF system, as a human free fast and reliable intra-operative cancer diagnosis method for guiding surgeon to clean the edges of cancerous tissues to be applied during the surgical operation, for pathological examinations. Fs-LIMS provides some unique diagnosis opportunities to investigate biochemical characteristics of cancerous tissues leading to obtain sensitive, fast, and reliable results. The analysis of tissue is based on distribution of molecular ion (m/z) peaks in low mass region (<m/z 100) in mass spectra. Fs-LIMS provides a great data for identification of tissues (healthy and cancerous) in details. The effect of laser pulse was investigated in this study and also different types of lasers are utilized for various investigations from surgery to spectroscopy. Methods: The experimental setup mainly consists of an ultrafast (90 fs) laser system, a mass spectrometer, laser-tissue interaction/ablation chamber, data collection, and analysis system with windows based fast digital-storage oscilloscope, statistical application codes which have been developed by our group for analyses running under MATLAB software. Results: Paraffin embedded colon tissues have been distinguished from each other with statistical approaches using fs-LIMS based data as an alternative to histological and pathological examinations. Conclusion: The fs-LIMS method provides a powerful novel approach to identify and analyze tissues. This promising method provides a fast and reliable (free of human mistakes) diagnosis and guidance for pathologists, surgeons, and patients during surgical operations, as well as increase the significance of mass spectrometric tissue analysis methods, especially with those capability of molecular identification of tissues. Lasers Surg. Med. © 2018 Wiley Periodicals, Inc.