Chemical signals are essential for communication between living organisms. Dogs possess two sensory organs enabling chemical communication; the main olfactory system and the vomeronasal organ (VNO). Besides, contact chemoreception is also pertinent, by which non-volatile molecules, including but not limited to proteins, are recognized as chemical signals. However, non-volatile chemical signals have been sparsely studied in dogs. Therefore, we aimed to examine the urinary proteins of female domestic dogs during estrus and anestrus phases to detect and identify such non-volatile chemical signals.