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Uterine contractions are important for various functions of the female reproductive cycle. Contractions are generated, in part, by electrical coupling of smooth muscle cells of the myometrium, the main muscle layer of the uterus. Aberrant myometrial electrical activity can lead to uterine dysfunction. To better understand and treat conditions associated with aberrant activity, it is crucial to understand the mechanisms that underlie normal activity. Here, we used microelectrode array (MEA) to simultaneously record and characterize myometrial electrical activities at high spatial and temporal resolution. Mouse myometrial longitudinal muscle tissue was isolated at different stages throughout the estrous cycle and placed on an 8×8 MEA. Electrical activity was recorded for 10 min at a sampling rate of 12.5 kHz. We used a spike-tracking algorithm to independently analyze each channel and developed a pipeline to quantify the amplitude, duration, frequency, and synchronicity of the electrical activities. Electrical activities in estrous were more synchronous, and had shorter duration, higher frequency, and lower amplitude than electrical activities in non-estrous. We conclude that MEA can be used to detect differential patterns of myometrial electrical activity in distinct estrous cycle stages. In the future, this methodology can be used to assess different physiological and pathological states and evaluate therapeutic agents that regulate uterine function., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)