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This study investigated the effects of demineralization on teeth and bones evaluated by fluorescence spectroscopy and micro energy-dispersive X-ray fluorescence spectrometry (μ-EDXRF) in rats. For in vitro study, 20 teeth of Wistar rats were removed and decalcified to evaluate fluorescence. For in vivo study, 10 female Wistar rats aged 6months were randomized into 2 groups: Control Group (C): non-ovariectomized rats; Ovariectomy Group (OV): ovariectomized rats to induce osteoporosis. The fluorescence spectroscopy of the teeth was performed for long-term (until 180days). For ex vivo study, the tooth and femur bone of the Wistar rats were removed at 180days to perform fluorescence spectroscopy using excitation laser at 408 and 532nm and μ-EDXRF for calcium (Ca) and phosphorus (P) analysis. There were no intergroup differences in fluorescence spectra with laser at 408nm (p≥0.05), but there were changes in the fluorescence spectra using laser at 532nm which led to both the wavelength shift and changes in the band area (p<0.05). The concentrations of P and Ca for the dentine and cortical bone, respectively, were significantly reduced in OV (p<0.05). Demineralization leading to loss of tissue quality may be assessed by fluorescence spectroscopy using 532nm laser. These findings corroborate those obtained by μ-EDXRF., (Copyright © 2016 Elsevier B.V. All rights reserved.)