Human arsenic metabolism produces a number of species with varying toxicities; the presence of some has been identified while the existence of others has been postulated through indirect evidence. Speciation methods for the analysis of arsenite (AsIII), monomethylarsonous acid (MMAIII), dimethylarsinous acid (DMAIII), arsenate (AsV), monomethylarsonic acid (MMAV), dimethylarsinic acid (DMAV), arsino-glutathione (As(GS)3), monomethylarsino-glutathione (MMA(GS)2) and dimethylarsino-glutathione (DMA(GS)) were developed in this study through the use of cation exchange and reverse phase chromatography in a complementary manner. Electrospray ionization mass spectrometry (ESI-MS) was used for molecular identification of the arsenicals while inductively coupled plasma mass spectrometry (ICP-MS) was employed for quantitation purposes. Validation of the developed methods against each other for the quantitation of trivalent and pentavalent arsenicals was performed. The effect of reduced glutathione (GSH) concentration on the formation of arsenic-glutathione (As-GSH) complexes was studied. In the presence of glutathione, the occurrence of chromatographic artifacts on the cation exchange column was observed. The stability of trivalent arsenicals and As-GSH complexes was studied at various pH conditions. The results shed light on the importance of sample preparation, storage and proper choice of analytical column for the accurate identification of the As species. Reinvestigation of some of the previously reported As speciation studies of glutathione-rich biological samples needs to be performed for the verification of occurrence of As-GSH complexes and DMAIII.