Clotrimazole is an azole anti-fungal medication used primarily in the treatment of a wide range of fungal dermal infections. Examples include thrush, athlete’s foot and ringworm. It has limited oral absorption and is commonly available in various topical preparations, including creams, lotions, and solutions. As well as its anti-fungal activity, clotrimazole has become a drug of interest in the treatment of sickle cell disease, malaria and some cancers. Clotrimazole has generic status and is thus globally produced and prescribed. It is on the World Health Organization’s (WHO) list of essential medicines, which serves as a model of the safest and most effective medications needed by a health system. This application brief describes the use of a PerkinElmer Quasar™ C18 (150 x 4.6 mm, 5 µm) column in comparison with a GL Sciences Inertsil ODS-3 (150 x 4.6 mm, 5 µm) column for the analysis of clotrimazole, conducted by a pharmaceutical company in Chile.