Abstract Identification of enzymes, regulators and transporters involved in different metabolic processes is the foundation to understand how organisms function. There are, however, many difficulties in identifying candidate genes as well as in proving their in vivo roles. In this review, we describe different approaches utilized in Arabidopsis thaliana to identify gene candidates and experiments required to prove the function of a given candidate. For example, we use the production of methionine-derived aliphatic glucosinolates that represent major defence compounds in A. thaliana. Nearly all biosynthetic genes, as well as the first sets of regulators and transporters, have been identified. An array of approaches, i.e. classical mapping, quantitative trait loci (QTL) mapping, eQTL mapping, co-expression, genome wide association studies (GWAS), mutant screens and phylogenetic analyses, has been exploited to increase the number of identified genes. Here we summarize the lessons learned from the different approaches used over the years with the aim to help designing and combining new approaches in the future.