It is rare in nature, having been only found in meteorites,[2] and some antibiotics of fungal origin, such as alamethicin and some lantibiotics.

In the laboratory, 2-aminoisobutyric acid may be prepared from acetone cyanohydrin, by reaction with ammonia followed by hydrolysis.

Katoh et al. used flexizymes[5] and an engineered a tRNA body to enhance the affinity of aminoacylated AIB-tRNA species to elongation factor P.[6] The result was an increased incorporation of AIB into peptides in a cell free translation system.

Iqbal et al.. used an alternative approach of creating an editing deficient valine—tRNA ligase to synthesize aminoacylated AIB-tRNAVal.

The aminoacylated tRNA was subsequently used in a cell-free translation system to yield AIB-containing peptides.