The parameters critically influencing the delivery rate on the biodegradable microsphere-mediated femtosecond (fs) laser perforation are investigated in detail with the aim of developing efficient molecular delivery. Cell membrane was perforated by the irradiation of weakly focused fs laser pulses to the spherical polylactic acid microspheres conjugated to the cell membrane. The delivery of fluorescein isothiocyanate-dextran and fluorescent silica particles to A431 cells is investigated in detail. The increase in the number of irradiated laser pulses had resulted in the increase of delivery rate. The delivery rate depends on the size and functionalization of fluorescent silica particles in which silica particles of 100 nm in diameter were able to be delivered into 20% of the irradiated cells, suggesting that the pore sizes are large enough for the delivery of therapeutic agents into cells. These findings contribute to the development of an efficient and safe phototherapy and drug delivery.