In this book, we demonstrate a new single cell optoporation and transfection technique using femtosecond Gaussian laser beam and optical tweezers. Tightly focused near-infrared (NIR) femtosecond laser pulse was employed to transiently perforate the cellular membrane at a single point of MCF-7 cancer cell. A distinct technique is developed by trapping the microparticle using optical tweezers to focus femtosecond laser exactly on the cell membrane to puncture it for the desired time. Subsequently, calculated amount of external gene was introduced in the cell by trapping and inserting same plasmid coated microparticle into optoporated cell using optical tweezers. Various experimental parameters such as femtosecond laser exposure power, exposure time, punctured hole size, exact focusing of femtosecond laser and cell healing time were closely analyzed to optimize the best conditions for cell viability. Trapped particle position is detected by another co aligned continuous wave laser. In the end, after insertion of plasmid coated microparticle, the targeted cells exhibited green fluorescent protein (GFP) under the fluorescent microscope hence confirming successful genetic change.