Cercosporin has excellent properties of photosensitization that have been widely used in organophotocatalyst and photodynamic therapy as well as an antimicrobial agent. Therefore, there is a need to quantify it accurately with accessible methods. A comparative analysis of cercosporin quantification obtained by spectrophotometry (SPEC) and high-performance liquid chromatography (HPLC) was performed for nineteen Cercospora coffeicola isolates from different coffee-producing municipalities in Brazil. Image analysis of cercosporin crystals was performed in isolates with either high or low production of the toxin. Our results show that SPEC and HPLC are equally valid for the cercosporin evaluation of C. coffeicola cultures grown in vitro. The isolates with high cercosporin production had a higher crystal number and size when compared to the one with low cercosporin production.