Abstract Background Although customized endonucleases [transcription activator-like effector nucleases (TALENs) and RNA-guided endonucleases (RGENs)] are known to be effective agents of mutagenesis in various host plants, newly designed endonuclease constructs require some pre-validation with respect to functionality before investing in the creation of stable transgenic plants. Results A simple, biolistics-based leaf epidermis transient expression test has been developed, based on reconstituting the translational reading frame of a mutated, non-functional yfp reporter gene. Quantification of mutation efficacy was made possible by co-bombarding the explant with a constitutive mCherry expression cassette, thereby allowing the ratio between the number of red and yellow fluorescing cells to serve as a metric for mutation efficiency. Challenging either stable mutant alleles of a compromised version of gfp in tobacco and barley or the barley MLO gene with TALENs/RGENs confirmed the capacity to induce site-directed mutations. Conclusions A convenient procedure to assay the cleavage activity of customized endonucleases has been established. The system is independent of the endonuclease platform and operates in both di- and monocotyledonous hosts. It not only enables the validation of a TALEN/RGENâ s functionality prior to the creation of stable mutants, but also serves as a suitable tool to optimize the design of endonuclease constructs.