The orange pigmentation of the ascomycete fungi Neurospora and Fusarium is mainly due to the accumulation of neurosporaxanthin, a carboxylic apocarotenoid whose possible biotechnological applications have not been investigated. From the discovery of the first enzyme of the biosynthetic pathway in 1989, the prenyltransferase AL-3, to the recent identification of an aldehyde dehydrogenase responsible for the last biosynthetic step, all the enzymes and biochemical reactions needed for neurosporaxanthin biosynthesis in these fungi are already known. Depending on the culture conditions and/or genetic background, Neurospora and Fusarium may produce large quantities of this xanthophyll and minor amounts of other carotenoids. This chapter describes methods for the growth of Neurospora crassa and Fusarium fujikuroi for improved neurosporaxanthin production, the analysis of this xanthophyll, its separation from its carotenoid precursors, and its identification and quantification.