This study aimed to develop a method to determine the degree of oocyte maturation in metaphase II in situ based on the balance between mitochondrial respiratory activity and lipid metabolism using resonance Raman spectroscopy. A decrease in the respiratory activity of overmatured oocytes was indicated by the reduced intensities of the resonance Raman bands corresponding to reduced cytochrome c in the cytoplasm. Moreover, the increased lipid concentration in overmature oocytes indicated lower lipid metabolism with a decreased mitochondrial function. New indexes were defined in terms of the ratios of the representative Raman peak intensities of reduced cytochrome c (750 and 1127 cm-1) to those of lipids (1438 cm-1 ) and they successfully classify the oocytes into groups based on their quality, which varied with their maturation degree. The high development rate of embryos that were fertilized in vitro after laser irradiation showed that laser irradiation was noninvasive to oocytes. The evaluation of two factors in situ, the active respiration and lipid metabolism, means to catch the most fundamental biochemical reactions of life activities. Our results demonstrate the potential application of resonance Raman spectroscopy as a new, noninvasive, and universal cell evaluation technology, for not only oocytes but also more general cells such as somatic cells and iPS cells.