The introduction of heterologous genetic information, particularly of multiple genes, into mammalian cells is a key technology in contemporary experimental biological research. The coexpression of fluorescently tagged sensors is required to simultaneously analyse multiple parameters in living cells and the coexpression of several proteins is necessary to manipulate cell fate in stem cell biology. Current technologies for multigene expression in mammalian cells are inefficient, inflexible and time-consuming. In this paper we describe MultiLabel, a novel and highly efficient modular plasmid-based eukaryotic expression system. Independent expression vectors are assembled by a Cre/LoxP reaction into a plasmid with multiple expression cassettes. MultiLabel enables rapid construction of multigene expression vectors for the single-step creation of transiently or stably transfected mammalian cells.