A novel mixed hemimicelle-based solid-phase extraction method using cetyltrimethylammonium bromide (CTAB) adsorbed onto the surface of magnetic Fe 3 O 4 nanoparticles was developed for extraction/preconcentration of ultra-trace amounts of indomethacin from biological fluids. Magnetic nanoparticles were synthesised via a simple chemical co-precipitation method and characterised by a transmission electron microscope, an X-ray diffractometer and Fourier transform infrared techniques. The method utilises the unique properties of Fe 3 O 4 nanoparticles including high surface area and superparamagnetism causing rapid separation (<20 min) and low adsorbent usage (only 50 mg). A comprehensive study on the parameters affecting the adsorption efficiency such as the amount of CTAB, pH of the solution, desorption conditions, amount of Fe 3 O 4 nanoparticles, extraction and preconcentration time, sample volume and ionic strength are presented. Under optimum conditions, the method's linearity was over a range of 25-450 ng ml −1. The limit of detection of 8.6 ng ml −1 , an enrichment factor of 99 and the relative standard deviation (%RSD) of 1.9% (for a concentration of 50 ng ml −1) were obtained. The method was successfully applied to determine indomethacin in human plasma and urine samples. Good recoveries (89-102%) with low % RSD, (1.7-2.7%) were achieved.