System and method for differentiating tissue margins in a biological sample using pulsed laser excitation and time-gated detection. A region containing a biological tissue is irradiated with substantially monochromatic pulsed laser light to thereby produce Raman scattered photons. The Raman scattered photons are detected using time-gated detection to thereby obtain a Raman spectroscopic image from the irradiated region characteristic of either a neoplastic portion or a non-neoplastic portion of the region containing the biological tissue. A boundary between a neoplastic portion and a non-neoplastic portion is differentiated and the boundary location in the Raman spectroscopic image is displayed.