The establishment of non‐invasive analytical tools for assessing the in‐situ use of biomaterials for surgical implants or scaffolds in tissue engineering and polymer‐based therapies is fundamental. This study established a method for fluorescent tracking of the degradation of a chitosan membrane scaffold for use in vitro in bioreactors and ultimately in vivo. The basis of this tracking system is a fluorescence emitting biomaterial obtained by covalent binding of the fluorophore tetramethylrhodamine isothiocyanate (TRITC) onto the backbone of chitosan. Using confocal microscopy, this study quantitated the reductions in fluorescence intensity of the membrane and correlated these decreases with weight loss during polymer breakdown, thereby providing a technique for non‐destructively assessing the extent of degradation of chitosan materials over time in vitro. Using multispectral imaging in a mouse model, the study assessed the degradation profile of the fluorophore‐labeled biomaterial in vivo in real time and identified the dispersing pathway of the chitosan membrane degradation products in vivo. The results revealed that TRITC conjugated chitosan was biocompatible and supported bone cell growth. The changes in fluorescence intensity correlated well with weight loss up to 16 weeks of in vitro culture and could be monitored over two weeks in vivo. Copyright © 2011 John Wiley & Sons, Ltd.