A species-specific isotope dilution analysis (SS IDA) method was developed for the first time for the determination of selenoprotein P (SEPP1) in human plasma/serum at the protein level by double affinity HPLC-ICP-MS. In this regard, a standard and a spike of SEPP1 were produced by cell-free E. coli protein synthesis, where Se (ICP tag) was introduced in the form of selenomethionine (SeMet) allowing for the absolute SEPP1 quantification by ICP-MS. A complete characterization of the standard and the spike was carried out in terms of isotopic composition and Se mass fraction by collision-cell ICP-MS to ensure SI-traceability. Method development and validation were conducted using the reference materials BCR-637, extensively analysed for its Se species, and the SRM 1950, which provides reference values for Se species (including SEPP1). Stability of the isotope ratio R_77/76 in the sample blends was tested for one month with negligible change. Relative expanded uncertainties of 5.7% and 7.7% were achieved for BCR-637 and SRM 1950 for mass fractions of 55.5 and 63.9 ng g⁻¹ Se for SEPP1, respectively. The developed SEPP1 SS IDA methodology could be a valuable tool to establish references values for selenoproteins in clinical chemistry and showed the potential of cell-free protein synthesis for the preparation of future stable isotope-labeled intact selenoproteins.