Alternative high throughput transfection methods are required to understand the molecular network of the cell, which is linked to the evaluation of target genes as therapeutic agents. Besides diagnostic purposes, the transfection of primary- and stem cells is of high interest for therapeutic use. Here, the cell release of trans- or exogene proteins is used to develop immune cancer therapies. The basic requirement to accomplish manipulation of cells is an efficient and gentle transfection method. Therefore, we developed an automatized cell manipulation platform providing high throughput by using GNOME laser transfection. Herein, the interaction of moderately focused laser pulses with gold nanoparticles in close vicinity to the cell membrane mediate transient membrane permeabilization. The exact nature of the involved permeabilization effects depends on the applied particles and laser parameters. Hereinafter, we describe investigations considering the parameter regime, the permeabilization mechanism and the safety profile of GNOME laser transfection. The experimental and calculated results imply a combined permeabilization mechanism consisting of both photochemical and photothermal effects. Furthermore, paramount spatial control achieved either by laser illumination with micrometer precision or targeted gold nanoparticle binding to the cells was demonstrated, allowing selective cell manipulation and destruction. Additionally, the possibility to manipulate difficult to transfect primary cells (neurons) is shown. These results give insights in the basic mechanisms involved in GNOME laser transfection and serve as a strong basis to deliver different molecules for therapeutic (e.g. proteins) and diagnostic (siRNA) use.