Introduction: There is a growing amount of evidence showing the significant analytical bias of steroid hormone immunoassays, but large numberof available immunoassays makes conduction of a single comprehensive study of this issue hardly feasible. Aim of this study was to assessthe analytical bias of six heterogeneous immunoassays for serum aldosterone, cortisol, dehydroepiandrosterone sulphate (DHEAS), testosterone,17-hydroxyprogesterone (OHP) and progesterone using the liquid chromatography coupled to the tandem mass spectrometry (LC-MS/MS).Materials and methods: This method comparison study included 49 serum samples. Testosterone, DHEAS, progesterone and cortisol immunoassayswere performed on the Abbott Architect i2000SR or Alinity i analysers (Abbott Diagnostics, Chicago, USA). DiaSorin’s Liaison (DiaSorin, Saluggia,Italy) and DIAsource’s ETI-Max 3000 analysers (DIAsource ImmunoAssays, Louvain-La-Neuve, Belgium) were chosen for aldosterone and OHPimmunoassay testing, respectively. All immunoassays were evaluated against the LC-MS/MS assay relying on the commercial kit (Chromsystems,Gräfelfing, Germany) and LCMS-8050 analyser (Shimadzu, Kyoto, Japan). Analytical biases were calculated and method comparison was conductedusing weighted Deming regression analysis.Results: Depending on the analyte and specific immunoassay, mean relative biases ranged from -31 to + 137%. Except for the cortisol, immunoassayswere positively biased. For none of the selected steroids slope and intercept 95% confidence intervals simultaneously contained 0 and 1,respectively.Conclusions: Evaluated immunoassays failed to satisfy requirements for methods’ comparability and produced significant analytical biases in respectto the LC-MS/MS assay, especially at low concentrations.