Up to now, the Fe content of nonmetallic particles has often been neglected in chemical evaluations due to the challenging analysis of matrix elements in nonmetallic inclusions (NMI) in steel by scanning electron microscope and energy dispersive spectroscopy analysis (SEM/EDS). Neglecting matrix elements as possible bonding partners of forming particles may lead to inaccurate results. In the present study, a referencing method for the iron content in nonmetallic inclusions in the submicrometer region is described focusing on the system Fe-Mn-O. Thermodynamic and kinetic calculations are applied to predict the inclusion population for different Fe/Mn ratios. Reference samples containing (Fe,Mn)-oxide inclusions with varying Fe ratios are produced by manganese deoxidation in a high-frequency induction furnace. Subsequent SEM/EDS measurements are performed on metallographic specimens and electrolytically extracted nonmetallic inclusions down to 0.3 µm. The limits of iron detection in these particles, especially for those in the submicrometric regime, as well as the possible influence of electrolytic extraction on Fe-containing oxide particles are examined. The measured inclusion compositions correlate well with the calculated results regarding segregation and kinetics. The examinations performed are reliable proof for the application of SEM/EDS measurements to evaluate the Fe content in nonmetallic inclusions, within the physical limits of polished cross-section samples. Only electrolytic extraction ensures the determination of accurate compositions of dissolved or bonded matrix elements at smallest particles enabling quantitative particle descriptions for submicrometric (particles ≤ 1 µm) steel cleanness evaluations.