A review of multidimensional separations such as comprehensive two-dimensional gas chromatography (GC x GC), comprehensive two-dimensional high performance liquid chromatography (HPLC x HPLC) and their applications in pharmaceutical and biological fields is presented with 71 references. A single CO2 cryo-jet loop modulator was developed for GC x GC and it can be used to modulate compounds higher than C6 effectively. Comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC x GC/TOF-MS) analyses of traditional Chinese medicine volatile oils such as Pogostemon cablin Benth (Cablin Patchouli), Forsythia suspensa (Thunb.) Vahl and Zedoary were reported also. As an emerging technology, multidimensional separations hold the promise and play an important role in the future pharmaceutical and biological fields.