The review article is devoted mainly to the description of applications of gold nanoparticles (GNPs) in separation sciences, especially in electromigration and chromatographic techniques. The applications of GNPs in particular separation methods, CE, microchip CE, MEKC, CEC, HPLC and GC, are classified according to the molecular size of the analytes from low-molecular-mass compounds via medium sized substances to biopolymers (proteins and nucleic acids). A very recent and promising utilization of GNPs for sample preparation, preconcentration and preseparation of selected analytes from complex matrices is presented as well. Moreover, in two introductory sections, typical preparation procedures of the GNPs and their modifications are presented and physico-chemical and analytical methods employed for characterization of the native and modified GNPs are briefly introduced.