While nowadays robotics enable performing whole genome functional screens within a few days, the availability of suitable cellular systems to investigate the function or pathway of choice represents as a major bottleneck. In most applications, it is desirable to use cell lines with stably inserted reporter systems or over expression/silencing of a target gene. We developed a simple recombination-based system, which allows serial introduction of genes/RNAi-constructs into cancer cell lines. This method yields highly standardized (isogenic) stable cell line libraries with hyperactivation or inactivation of a gene of choice in a constitutive or tetracycline-inducible fashion. We also provide proof-of-principle that this technique can be used for the construction of double recombinant cell lines, which allows for analyses at advanced levels of complexity, e. g. by the construction of double reporter systems.