A new sample preconcentration technique known as in-situ suspended aggregate microextraction (iSAME) was used for the extraction of acetaminophen, ibuprofen, indomethacine, ciprofloxacin and diclofenac in river waters. The iSAME has been optimized in terms of surfactants selection and concentration, sample volume and extraction time. A combined surfactant of hexadecyltrimethyl ammonium bromide (CTAB) with 5-sulfosalicylic acid dihydrate (SSA) was studied to attain better extraction recoveries for the targeted pharmaceuticals. The separation of analytes was performed using capillary electrophoresis (CE) with online preconcentration by field enhancement sample injection (FESI) mode. The linearity, detection limits and recoveries were recorded and the sensitivity increased with enhancement factor of 11–140 folds. The developed method was successfully applied for the detection of selected pharmaceuticals in river water samples.