Isotope dilution techniques have been widely applied in research investigating the fate and availability of different elements in soils. Typically, radioactive isotopes of metals and metalloids have been used to measure the bioavailable pool of metals in soils (L values). For Cu, the radioactive isotope procedure is impossible for determining the bioavailable pool of Cu in soils (L values), owing to the short half-life of the ⁶⁴Cu radioisotope (12.4 h), which makes access and handling very difficult. However, more recently, rapid and sensitive analytical techniques such as ICP-MS have led to the use of stable metal isotopes. Therefore, we developed a technique using enriched ⁶⁵Cu stable isotope and measurement of ⁶³Cu/⁶⁵Cu ratios by quadrupole inductively-coupled plasma mass spectrometry (ICP-MS) to measure L values by plants grown in ⁶⁵Cu-spiked soils. Mass spectral interferences in detection of ⁶³Cu/⁶⁵Cu ratios in soil extracts and plant digests were found to be minimal. Isotope ratios determined by quadrupole ICP-MS compared well to those determined by high-resolution (magnetic sector) ICP-MS. The ⁶³Cu/⁶⁵Cu ratios in soil extracts and in plants (tomato and ryegrass) are identical and good agreement between the isotopically exchangeable Cu (E value) and bioavailable pool (L value) of Cu in soils has been found for a given soil, which suggests the isotopically exchangeable Cu is identical to the potentially bioavailable Cu in soils. The theory of isotope dilution, the procedures of L value measurement and factors affecting the determination of L values are also discussed.