The complementary use of LC-ESI-MS and LC-ICP-MS for characterization of native and recombinant copper proteins (molar mass range 10–20 kDa) was investigated. SEC and IC separation protocols were implemented for hyphenated ICP-MS analysis. The studies showed that validation of the methods addressing metal integration on a quantitative basis via metal to sulfur ratios demanded complementary determinations of molar mass. Reversed phase LC-ESI-TOF-MS measurements showed point mutation for the investigated recombinant apo-plastocyanin. Moreover, both recombinant proteins, i.e. plastocyanin and the Cu_A domain of cytochrome c oxidase from the cyanobacterium Synechocystis, lost their N-terminal amino acid upon expression. Since in both cases methionine formed the N-terminus the theoretical metal to sulfur ratio of the protein was changed. Excellent precision ranging at 3 ppm (N = 5) could be achieved for the determination of multiply charged ion patterns by LC-ESI-TOF-MS. The precision of the molar mass determination after deconvolution ranged at 5–15 ppm (N = 5). The intact metal containing copper proteins were measured by flow injection-ESI-TOF-MS under non-denaturing conditions (pH 5). Mass accuracy of ESI-TOF-MS allowed confirming not only stoichiometry of metal ligation but also the oxidation state of the metal center in plastocyanin and the Cu_A domain of cytochrome c oxidase. Moreover, IC-ICP-MS measurements on isotopically enriched Cu proteins were accomplished showing the potential of hyphenated ICP-MS analysis in future tracer studies.