High-performance liquid chromatography is a frequently used analytical method in the analysis of pharmaceuticals, biomolecules, polimers and various organic and inorganic compounds. For a proper use of HPLC, it is essential to optimise the system for analysed analytes and to use detector sensitive to analytes for which it is crucial to understand the characteristics of the analytes. One of the applications of the HPLC method is the analysis of neurotransmitters and their metabolites in lizard brains. Most frequently analysed neurotransmitters and their metabolites are serotonin, dopamine, noradrenaline and adrenaline. Analyses which demonstrate localization and concentration of neurotransmitters and their metabolites assist in the comprehension of their roles in the central nervous system and brain circuits in which they operate or help with unraveling of brain evolution and evolution of brain functions with the lizard brain as an evolutionary model. Some of dopamine roles are in movement, endocrine regulation, motivation, learning, cognition and affective behaviour. Noradrenaline and adrenaline, except numerous roles as hormones in the rest of the body, in the central nervous system have roles in state of arousal, vigilance and alertness. Roles of serotonin are in mood, anxiety, fear, aggression and sexual behaviour. HPLC methods used for neurotransmitter measurement are HPLC/UV, HPLC/MS, HPLC/FLD and most frequently used HPLC/ECD. With the use of HPLC methods when analysing lizard brains, neurochemical explanation of previously observed lizard behaviour is provided.