Oocyte energy metabolism is important for its developmental capacity and the nucleotides ATP, ADP and AMP play critical roles as energy currency of biological reactions. During the early stages of oocyte maturation, cyclic AMP (cAMP) is hydrolysed to AMP which we hypothesize can be recycled through the adenosine salvage pathway to ADP and then ATP, as an alternate energy generating pathway. Significant changes in the amount of these nucleotides may also impact oocyte energy charge (EC), an index used to measure a cell’s energy status through the assessment of the energy stored in its adenylate system. To facilitate the study of oocyte adenosine nucleotide metabolism, we developed a highly selective and sensitive LC-MS/MS method for the simultaneous quantification of low nanomolar to micromolar concentrations of adenosine, ATP, ADP, AMP, and cAMP. Metabolites were separated on a porous graphitic carbon column, which offered superior retention and chromatographic resolution, however rigorous conditioning protocols were required to ensure repeatability. The method was validated using αMEM culture media containing 0.3% BSA as sample matrix. The method was linear from 5nM to 10µM for all analytes with correlation coefficients above 0.998. The recovery ranged from 77% to 107% from 20nM to 5µM. Precision (%CV) was below 15% from 50nM. The limits of detection and quantification were 5nM and 10nM, respectively. The method was successfully applied to quantify nucleotides in COV434 granulosa cell conditioned media and cell extract. The mitochondrial uncoupler CCCP decreased nucleotide levels in media 2-5-fold, and cAMP modulators forskolin and IBMX increased cAMP secretion 14-fold. Nucleotide concentrations from these samples ranged between 5-90nM. Simultaneous quantification of ATP, ADP and AMP enabled calculation of the EC. Mouse oocyte EC was 0.88-0.92 throughout maturation, indicating that the EC is buffered. This novel mass spectrometry method will allow detailed interrogation of energy generating systems in oocytes.