Biogenic amines (BAs) are low molecular weight nitrogenous compounds, formed by the breakdown of proteins in highly perishable food products such as fish. BAs can affect human health and are associated with cases of food poisoning. The formation of Bas such as histamine, putrescine, and tyramine were determined, via Process Analytical Chemistry (PAC), in three species of freshwater fish available in markets of city of Pasto in southern Colombia: rainbow trout, tilapia, and cachama. We evaluated the formation of BAs during the fish conservation processes and considered a multifactorial design with two levels. The factors studied were: fish species, slaughter type, storage temperature, and time to purchase. Out of the three fish species studies, tilapia samples revealed the highest average content of putrescine and histamine, with values of 5.4 µg/g and 10.04 µg/g, respectively. Tyramine was not detected in any of the experiments performed. The observed values of BAs in the samples analyzed were below locally tolerated maximal values and the European standard (200 µg/g). However, their presence reveals that factors such as sample storage temperature and time to consumption triggered their formation.