Abstract The knowledge of the speciation of elements at trace and ultra-trace level, in biological and environmental media is essential to acquire a better understanding of the mechanisms of toxicity, transport and accumulation in which they are involved. Determining the speciation of an element in a given medium is challenging and requires the knowledge of different methodological approaches: the calculation approach and the experimental approach through the use of dedicated analytical and spectroscopic tools. In this framework, this mini-review reports the approaches to investigate the speciation of elements in biological and environmental media as well as the experimental techniques of speciation analysis, illustrated by recent examples. The main analytical and spectroscopic techniques to obtain structural, molecular, elemental and isotopic information are described. A brief overview of separation techniques coupled with spectrometric techniques is given. Imaging and micro-localisation techniques, which aim at determining the in situ spatial distribution of elements and molecules in various solid samples, are also presented. The last part deals with the development of micro-analytical systems, since they open crucial perspectives to speciation analysis for low sample amounts and analysis on field.