Acanthamoeba is an opportunistic pathogen which is the causal agent of several human infections such as Granulomatous Amoebic Encephalitis, Acanthamoeba keratitis and other disseminated infections. Furthermore, current therapeutic measures against Acanthamoeba infections are arduous, and show limited efficacy against the cyst stage of Acanthamoeba. There is a pressing need to search and evaluate new therapeutic agents against these protozoa. Our approach for evaluating possible new drugs is an initial in vitro screening assay based on general metabolic activity of the cells. In this study we compare two agents, AlamarBlue® and PrestoBlue® for this initial screen. Both reagents can be used to indicate metabolism by changes in their absorbance or fluorescence. The assay is carried out in a 96-well plate format and fluorescence can be measured after an inoculation period of as little as 10. min, but more typically 96. h. This to the best of our knowledge this is the first time that both compounds are directly compared using absorbance and fluorescence measurement. We conclude that for the specific case of Acanthamoeba both agents AlamarBlue® and PrestoBlue® are equally useful to determine cell viability.