The key endogenous fluorophores in tissues are collagen, elastin, Nicotinamide adenine dinucleotide, keratin, melanin and hemoglobin which are extensively being investigated using LIF. Collagen being the major component of the extracellular matrix plays a key role during tissue remodeling, which if monitored noninvasively, will provide an opportunity to assess the wound healing progression and hence will help in the planning of subsequent treatment. The prospective of second harmonic generation microscopy as a non-invasive imaging tool to monitor collagen near the wound boundaries during tissue regeneration is very well documented. Likewise, Raman spectroscopy and histological techniques have also been employed to evaluate native collagen in wounds. The basic purpose of adopting LIF here is to monitor collagen non-invasively during tissue regeneration by measuring the corresponding autofluorescence and testing the performance of the technique. The actual motivation for the present work came from the outcome of our previous study, wherein, for the first time we demonstrated the usefulness of ex vivo autofluorescence in assessing endogenous collagen in granulation tissue during the wound healing progression following laser therapy. In that study, the wound granulation tissue shown a gradual increase in the collagen fluorescence during all the post-wounding days along with the microscopic evidence of collagen deposition as shown by the quantitative scoring of the histological features highlighting the capability of the technique in collagen monitoring. Since collagen staining by Masson’s trichome stain used in that report had limitations of being unable to differentiate between different types of collagen fibers, and failed to differentiate between thin elastin and collagen fibers. To overcome these, we have utilized Picro-Sirius red in the present study for the identification of different collagen fibers along with the information on their occurrence and orientations. In the present study, in addition to the qualitative scoring of the Picro-Sirius red stained histological sections for thicknes, occurrence and orientation, a quantitative assessment for total collagen deposition in the tissue was also perfomed using image analysis along with its comparision with spectroscopic findings of endogenous collagen. Histological sections were examined in a blinded fashion and scored qualitatively for the thickness of the collagen fiber, their occurrence and orientations by an expert pathologist. The scores for different histological parameters of un-wounded control, unilluminated control and laser treatment groups were listed separately in tables 2, 3 and 4 respectively. Presently available methods of examining collagen in biological tissues are histochemistry and immunohistochemistry wherein both techniques involve several steps in tissue processing and it introduce undesirable structural variations in the collagenous matrix.