β-carotene is an increasingly sought-after organic pigment with antioxidant properties and a vitamin precursor. Yarrowia lipolytica, though unable to naturally synthesize carotenoids, can produce high amounts of the precursor acetyl-CoA making it a promising host for metabolic engineering towards novel biotechnological production of carotenoids. Here, we describe a synthetic biology methodology for Y. Lipolytica metabolic engineering based on Golden Gate DNA assembly for the generation of a multigene cassette, subsequent transformation enabling β-carotene biosynthesis, and quantification of the compound.