We describe here a competitive hybridization assay using TRACE technology which can be used for realtime monitoring of oligonucleotide hybridization. This assay quantifies all kinds of oligonucleotides in biological fluids without extraction. The assay makes use of two different probes and involves a fluorescent transfer process. As fluorescence measurements are not destructive, they can be sequentially repeated, thereby allowing comparison of the hybridization kinetics and binding strength of chemically modified backbone oligonucleotides (>0.5 nM) in biological media. The assay was validated for pharmacokinetic analysis of phosphodiester and phosphorothioate oligonucleotides in plasma and in different organs (liver, kidneys, lungs, spleen) at low concentrations (0.4 mg/kg, corresponding to clinical doses). Respective sensitivities for phosphodiester and phosphorothioate were 0.2 and 0.8 pmol/ml in plasma and 2 and 8 pmol/g in tissues, which allow to recover intact phosphorothioate sequences in some organs even after 24 h.