Pattern separation is the process of transforming highly similar sensory inputs into distinct, dissimilar representations. It takes place in the hippocampus and is thought to be used in episodic memory. Impaired pattern separation performance has been recognized as a predictor for the development of cognitive impairments such as dementia in humans and as being present in patients with schizophrenia and post-traumatic stress disorder (PTSD). In this protocol, we describe how to implement a simple and robust object pattern separation (OPS) task in mice and rats that we have previously established and validated. This two-trial memory task uses specific object locations so differences in performance can be calibrated with the extent of object movement. Changes in performance are indicative of spatial pattern separation. In contrast to other pattern separation tasks, the OPS task allows detection of spatial pattern separation performance bidirectionally. Furthermore, the OPS task is cheaper and easier to use and interpret than other tasks that use more than two objects or that are touch-screen based. The entire protocol, from vivarium acclimatization to training of the animals, takes ~35-41 d. After successful training, the animals can be tested repeatedly, and three OPS experiments (n = 20-24 per experimental day) can be performed per week. A standard level of expertise in behavioral studies in rodents is sufficient to successfully integrate this paradigm into an existing rodent test battery.