Superparamagnetic iron oxide nanoparticles (SPION) are very promising contrast media, especially for molecular imaging, due to their superior NMR efficacy. They even have wider biomedical applications such as in drug and gene delivery, tissue engineering and bioseparation, or as sensitive biological nanosensors. By coupling them to affinity ligands, SPION can bind to drugs, proteins, enzymes, antibodies or nucleotides. For in vitro biomedical applications, the detection of molecular interaction is possible by using a diversity of systems capable of sensing the magnetic properties of these materials. The goal of the present work was to develop and validate various in vitro biomedical applications of ultrasmall superparamagnetic particles of iron oxide (USPIO), including some that are not related to their magnetic properties. USPIO coated with dextran, starch or bisphosphonate exposing carboxylate groups were synthesized and some of them were functionalized by conjugating various biomolecules, such as biotin, streptavidin and apoptosis, or VCAM-1 specific peptides. The in vitro biomedical applications assessed in the present work included: (1) the relaxometric measurement of antibody concentration, cell receptor expression, molecular interaction, and enzymatic activity in aqueous suspensions; (2) MRI visualization of cells and detection of molecular interaction in an ELISA system; (3) ELISA applications of USPIO derivatives; and (4) detection of specific biomolecules by histochemistry. Our results confirm that rapid and simple in vitro detection of a diversity of functionalized SPION with relevance in medicine is possible by the existing NMR techniques and by chemical staining reactions. The protocols can be applied to minimally prepared biological samples (e.g. whole blood, blood plasma or serum, cell suspensions, biopsies, histological preparations, etc.), and often do not need complicated systems of signal amplification. The use of SPION labeled compounds could furthermore contribute to cost reductions in the diagnosis and in patient care. Copyright © 2010 John Wiley & Sons, Ltd.