Synthetic quorum-sensing systems in mammalian cells has enabled the implementation of time- and distance-dependent bioprocesses, as well as the design of synthetic ecosystems emulating clinically important host-parasite interactions. In this chapter, we provide a detailed protocol of the design of a mammalian cell-to-cell signaling device and its integration into a mammalian quorum-sensing system for cell density-induced expression product genes. Cell-to-cell signaling is based on a sender cell, metabolically engineered for expression of alcohol dehydrogenase converting ethanol into acetaldehyde, and a receiver cell line for the dose-dependent translation of the acetaldehyde concentration into transgene expression by an acetaldehyde-responsive promoter. This protocol can be adapted easily to various cell types and transgenes for the design of versatile mammalian cell-based quorum-sensing systems.