Several cell transfection techniques have been developed in the last decades for specific applications and for various types of molecules. In this context, laser based approaches are of great interest due to their minimal invasiveness and spatial selectivity. In particular, laser induced plasmon based delivery of exogenous molecules into cells can have great impact on future applications. This approach allows high‐throughput laser transfection by excitation of plasmon resonances at gold nanoparticles non‐specifically attached to the cell membrane. In this study, we demonstrate specific gene‐knockdown by transfection of Morpholino oligos using this technique with optimized particle size. Furthermore, we evaluated the cytotoxicity of plasmonic laser treatment by various assays, including LDH activity and ROS formation. In summary, this study gives important insights into this new approach and clearly demonstrates its relevance for possible biological applications. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)