Lolitrem-B is a neurotoxic substance that causes the ryegrass staggers syndrome in cattle, sheep, and horses consuming ryegrass (Lolium perenne L.) infected with endophytic fungus Neotyphodium lolii (Latch, Christensen, and Samuels) Glenn, Bacon, and Hanlin. Due to the increasing use of ryegrass pasture in southern Chile, it is necessary to know the content of lolitrem-B to optimize grazing management and reduce the effect on animal health. The objective of this study was to implement a method to determine lolitrem-B in ryegrass. Ryegrass samples were used without the endophyte, freeze-dried, and kept in darkness at -18 °C to establish the method. Then, a pure lolitrem-B standard was added and the toxin was extracted with a mixture of chloroform-methanol (2:1), purified in manually prepared silica gel 60 columns. Five standard addition levels were studied (0.05, 0.10, 0.30, 0.60, and 1.00 mg kg-1) with five replicates per level. The recovery was 96.6 to 99.9%, on average and the coefficient of variation (CV) ranged between 0.9 and 5.9%. Quantification was done by high-performance liquid chromatography (HPLC) and fluorescence detection. The quantification limit was 0.05 mg kg-1. This method of determining neurotoxin lolitrem-B in ryegrass samples was implemented for the first time in Chile. It is fast and inexpensive; it has good precision, accuracy and repeatability.