For over 30 years, the orange spots of male guppies have been an iconic example of carotenoid-based ornamentation. The humble guppy was the first of many model species used to study carotenoids in sexual coloration and it is a favoured text book example of signal evolution. It is therefore rather surprising that few have attempted to identify and quantify the carotenoids present in guppies. With some exceptions (most notably Greg Grether's work), researchers have instead tried to infer pigment concentration from colorimetric techniques such as reflectance spectrophotometry or photography. Three major obstacles exist for accurate biochemical determination the carotenoids present in guppies. First, the small body size means that only miniscule amounts are available for analysis. Second, fish skin carotenoids are esterified and samples therefore require potentially destructive hydrolysis. Third, some of the carotenoids are structurally similar, which poses a challenge for successful separation. We have developed a method to identify and quantify the individual carotenoids in guppy skin by using mild saponifiction followed by normal phase high performance liquid chromatography (NP-HPLC). It provides effective hydrolysis of the carotenoid esters without damage to the pigments and is sensitive enough to quantify the carotenoids in a single skin spot. We also used different photographic techniques to measure coloration of both free-swimming and sedated male guppies to investigate how accurate photographic methods are in estimating carotenoid levels.