Progress in natural product chemistry has always been strongly linked to innovations in analytical technology. The characterisation of metabolites in complex mixtures requires sophisticated techniques, which should provide good sensitivity and selectivity as well as structural information on the constituents of interest. This review outlines the most important chromatographic and spectral techniques which have been introduced in the field of natural products. Although there has been a very rapid evolution of methods over the last 50 years, the introduction of high-throughput screening programmes require even more efficient and sensitive methodologies which yield adequate on-line information for metabolite structure determination.