The possibility of fabricating micrometric pore size membranes is gaining great interest in many applications, from studying cell signaling, to filtration. Currently, many technologies are reported to fabricate such microsystems, the choice of which depends strictly on the substrate material and on the final application. Here, we demonstrate the capability with a single femtosecond laser source and experimental setup to fabricate micromembranes both on polymeric and multilayer metallic substrate, without the need for moulds, mask, and complex facilities. In particular, the flexibility of laser drilling was exploited to obtain microfilters with pore size of 8 and 18μm in diameter,on metallic and polymeric substrate, respectively, and controlled distribution. For evaluating the possibility to use such laser-fabricated membranes into biological assay, their biocompatibility has been investigated. To this aim, as a proof of concept, we tested the two materials into viability tests.The culture of mammalian cells on these microfabricated membranes were studied showing their compatibility with cells.