Giardia duodenalis is a cosmopolitan flagellated protozoan parasite with a wide range of hosts, including both domestic and wild animals, as well as humans. It reproduces in the small intestine causing giardiosis, a disease characterized by diarrhea, abdominal cramps, weight loss, and malabsorption. The aim of this study is to standardize the colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method for in vitro assays against G. duodenalis trophozoites. The MTT has been shown to be useful in determining active products against various biomodels and a good substitute for microscopic counting in determining parasite viability. To adapt this method to G. duodenalis, a series of assays were performed to optimize the test, starting with growth curves to observe the behavior of the protozoan cultures. Multiple assays were then performed, modifying certain variables such as initial inoculum, culture phase, or reagent concentrations, resulting in an effective standardized protocol. The results of this study can be of great utility in identifying compounds with activity against G. duodenalis.