Recently there has been concern regarding the use of flunitrazepam and other low-dose benzodiazepines in drug-facilitated sexual assault. These compounds are placed in drinks of unsuspecting victims and produce a sedative effect with anterorgrade amnesia. Chip-based microfluidic systems can provide a quick and disposable procedure for the detection of flunitrazepam and other nitrated benzodiazepines used in these crimes. This paper describes the application of indirect quenching of cyanine dye (Cy5) for detection of nitrated benzodiazepines. The separation is performed on a microfluidic device with a separation channel 8 cm long and 50 microm wide and utilizes indirect fluorescence detection with 635 nm laser excitation. The optimization of the separation using micellar electrokinetic chromatography with organic modifiers is described. A borate buffer containing 2.6 microM Cy5 dye, 15 mM sodium dodecyl sulphate (SDS) and 20% methanol is used. Complete separation of four target drugs occurs in under 2 min with limits of detection in the low microg/ml range. Overall the method provides a rapid and simple analysis for the presence of nitrated benzodiazepines in beverages and other similar preparations.