The Re-Os isotopic system is currently limited as a chronometer because of the lack of accurate gravimetric standards for Os spike calibration and uncertainty of the 187Re half-life, which is also dependent on the accuracy of Os spike calibration. We demonstrate that the concentration of an Os spike can be calibrated accurately to ±0.2%. The accuracy of this calibration was achieved by using a high-purity, stoichiometric, and anhydrous K2OsCl6 standard. We show that (NH4)2OsCl6, the standard material used by most other laboratories, is less reliable for Os spike calibration because it is hygroscopic and decomposes during heating. Nebulization and ionization in a plasma at 6500 to 8000 K does not discriminate Os of different oxidation states and chemical forms, allowing Os isotopic ratios in spike-normal mixtures to be equilibrated “on-line” and reproduced at the ∼0.02% level. Multiple isotope dilution measurements reproduce Os concentrations at the 0.04% level. The Os isotopic compositions of the standards determined by multiple collector inductively coupled plasma mass spectrometry are in excellent agreement with that of negative thermal ionization mass spectrometry. A strong linear relationship between instrumental mass fractionation factors (β) for Os and Ir in MC-ICP-MS allows us to determine the isotopic composition of the Os spike with high precision and accuracy. Application of such an accurately calibrated spike is anticipated to reduce the uncertainty of 187Re half-life significantly, thereby increasing the accuracy of the 187Re-187Os chronometer.