Many decades have seen the exploration of a novel methodology for developing microstructure in Microfluidic devices. Despite many methodologies, femtosecond laser technique has been a rising method in the conspicuous way of developing microstructures through direct write method which provides an ease of fabrication. For fabrication precise microstructure in semiconductor devices such as silicon, the femtosecond laser is preferred. An array of micro-holes has been created in monocrystalline silicon (100) using femtosecond laser ablation with multiple pulses. An investigation is carried out for obtaining the minimum diameter holes by varying the power for multiple pulses. The fabricated microhole array find its application in cancer cell separation from human blood. © 2018, Springer-Verlag GmbH Germany, part of Springer Nature.