AbstractTerritoriality is a form of social dominance concerning the use of space that ensures the territory owner primary access to critical resources. The territory is defended with visual displays, advertisement calls, physical attacks, or chemical signals. The territory is frequently estimated by mapping locations where an animal is observed engaging in territorial behavior or by tracking. However, these approaches may over- or underestimate the areas defended. Thus, the use of approaches explicitly determining defended areas is critical to properly characterize the territory. Intrusion experiments can elicit a response in territory holders, allowing one to characterize their aggressive responses; however, the aggressive response depends on the species. We describe an approach to experimentally estimate the territory size using playback experiments in a species that exhibits a stereotypical phonotactic response: the nurse frog, Allobates aff. trilineatus and develop a new behavioral index that allows assessing territory size in response to playbacks for a species with non-stereotyped phonotactic response: the endangered Lehmann’s poison frog, Oophaga lehmanni. We conducted 772 playback experiments on 18 males of A. aff. trilineatus, and 222 on nine males of O. lehmanni. We analyzed the results of playback experiments with three different area estimators regularly used to estimate space use and evaluated whether these estimates are correlated. The shape and size of territories varied among individuals and estimators in both species. Although we found that the absolute size of the territory depends on the method used, estimates were strongly correlated, meaning that different estimators similarly describe variation in territory size among males. Choosing an analysis method may not be particularly important for studying the characteristics of territoriality over space and time but using a systematic and standardized experimental approach that also incorporates the particularities of the aggressive response of each species is essential to understand the evolution of space use by poison frogs and other territorial species.