The development of robust methods for determining the concentration and speciation of antimony (Sb) in natural samples is essential to understanding its distribution and cycling in nature. Here we discuss our experiences with a variety of approaches for measuring the content and speciation of Sb in environmental matrices. Total Sb concentration measurements in waters require digestion with HNO3-HCl to release Sb from particulate material and may require a preconcentration step to remove Sb from saline matrices or to obtain the required sensitivity. Plant analyses require the use of HNO3-HBF4 or HNO3-HF while sediments require the use of HNO3-HCl to solubilise Sb and prevent adsorption to silicates. Methods for Sb speciation should be fit for purpose. Volatile Sb species can be measured successfully using SPME-GCMS, waters via hydride generation-trapping ICPMS and sediment extracts using HPLC-ICPMS. Extraction of Sb from sediments and plants presents a challenge; however, the use of citrate is adequate for extraction of Sb from sediments predominately containing Sb associated with Fe-Mn oxyhydroxide phases. We have been unable to successfully quantify organic Sb species in plants because of the oxidation of Sb(iii) to Sb(v) and the formation of artefact Sb species. The analysis of solid samples using X-ray absorption spectroscopy should be considered as it has been shown to discriminate between Sb(iii) and Sb(v) as well as Sb minerals, oxides and adsorbed Sb species.