We report two routes of chemical synthesis of arsinothricin (AST), the novel organoarsenical antibiotic. One is by condensation of the 2-chloroethyl(methyl)arsinic acid with acetamidomalonate, and the second involves reduction of the N-acetyl protected derivative of hydroxyarsinothricin (AST-OH) and subsequent methylation of a trivalent arsenic intermediate with methyl iodide. The enzyme AST N-acetyltransferase (ArsN1) was utilized to purify L-AST from racemic AST. This chemical synthesis provides a source of this novel antibiotic for future drug development.