Background: Multi functional reporter constructs are of great interest in molecular biology. For example such constructs are essential for development and target validation in high throughput screening. Current technologies apply the use of IRES elements. The main drawback of IRES constructs is the variable ratio of expression of the reporter genes. Methods: To overcome the current disadvantages in technology we make use of the self cleavage peptide T2A. To exemplify the feasibility of this approach we designed a tri-functional reporter construct coding for the firefly luciferase gene, a fluorescent protein gene and the puromycin resistance gene. These proteins are linked through the viral T2A peptide. Results: We were able to generate a multi functional reporter construct. This protein is translated in a single open reading frame and after translation the polyprotein is processed into three individual proteins using the viral T2A peptide. Via the FRT recombinase system we could generate a stable isogenic human cell line. Conclusions: Here we established a rapid system to generate stable cell lines expressing a tri-functional reporter construct. In contrast to constructs containing IRES elements our reporter is expressed in equimolar ratio because of the T2A processing. With the help of functional validation we could show that the processing of the polyprotein is highly efficient. The FRT system gives an interesting tool to rapidly generate isogenic cell lines.