Photoporation with free photothermal nanoparticles (NPs) is a promising technology for gentle delivery of functional biomacromolecules into living cells, offering great flexibility in terms of cell types and payload molecules. However, the translational use of photoporation, such as for transfecting patient-derived cells for cell therapies, is hampered by safety and regulatory concerns as it relies on direct contact between cells and photothermal NPs. A solution is to embed the photothermal NPs in electrospun nanofibers, which form a substrate for cell culture. Here we present a protocol for photothermal electrospun nanofiber (PEN)-mediated photoporation that induces membrane permeabilization by photothermal effects and enables efficient intracellular delivery of payload molecules into various cell types. By incorporating photothermal NPs within biocompatible electrospun nanofibers, direct cellular contact with NPs is avoided, thus largely mitigating safety or regulatory issues. Importantly, PEN photoporation is gentler to cells compared with electroporation, the most commonly used physical transfection method, resulting in higher-quality genetically engineered cells with better therapeutic potential. According to this protocol, it takes 2-3 d to prepare PEN culture wells with the desired cells, 3-4 d to optimize PEN photoporation parameters for intracellular delivery of payload molecules into different cell types in vitro and 4-5 weeks to evaluate the in vivo therapeutic efficacy of PEN-photoporated T cells. The protocol also provides details on how to construct the laser-based setup for performing photoporation experiments.