Purpose: To develop and validate a novel in-vivo phosphorus-31 magnetic resonance spectroscopy (31P-MRS) method to determine the absolute concentrations of phosphocreatine [PCr], inorganic phosphate [Pi], and adenosine triphosphate [ATP] in the vastus lateralis muscle. Materials and Methods: An external 6 mL plastic vial with 850 mM of methylenediphosphonic acid (MDP), fixed to the center of a commercial dual-tuned transmit/receive surface coil, was used to calibrate metabolite concentrations from spectral areas. A 15cm diameter, 4 L cylindrical phantom (35 mM H3PO4) was scanned on a custom coil holder using the same parameters. Reproducibility of the 31P-MRS measurements was determined in volunteers (2M/3F, age = 39.2±21.9 years) while accuracy was determined using phantoms of known concentrations. Eight young subjects (24.5+4.2 years; 4M/4F) and eight older subjects (59.6+4.5 years; 4M/4F) were scanned. Student’s t-test was used to compare older versus younger subjects. Results: The percent error between the calculated and known molarity of phantoms was 3.3±1.9%. The mean coefficient of variation for the measurements of [PCr] was 5.2±3.7 %. Phosphorus metabolite concentrations, including [PCr] (25.2±3.4 mM vs. 28.5±3.4 mM, p < 0.005), [ATP] (6.68±0.84 mM vs. 7.71±0.61 mM, p<0.05) and [Pi] (3.18±0.46 mM vs. 2.56±0.55 mM, p<0.05) were significantly lower in older versus younger subjects. A significant, negative correlation was found between [PCr] and BMI (r = -0.50, p < 0.05). Conclusion: Quantitative 31P-MRS measurements reveal previously unappreciated differences in skeletal muscle phosphorus metabolite concentrations between young and older subjects and may provide unique insights when combined with other metabolic tests.