Mycoplasma contamination in mammalian cell cultures is often overlooked yet is a serious issue which can induce a myriad of cellular changes leading to false interpretation of experimental results. Here, we present a simple and sensitive assay to monitor mycoplasma contamination (mycosensor) based on degradation of the Gaussia luciferase reporter in the conditioned medium of cells. This assay proved to be more sensitive as compared to a commercially available bioluminescent assay in detecting mycoplasma contamination in seven different cell lines. The Gaussia luciferase mycosensor assay provides an easy tool to monitor mammalian cell contaminants in a high-throughput fashion.