The fabrication details to form large area systematically changing multishape nanoscale structures on a chip by laser interference lithography (LIL) are described. The feasibility of fabricating different geometries including dots, ellipses, holes, and elliptical holes in both - and - directions on a single substrate is shown by implementing a Lloyd’s interferometer. The fabricated structures at different substrate positions with respect to exposure time, exposure angle and associated light intensity profile are analyzed. Experimental details related to the fabrication of symmetric and biaxial periodic nanostructures on photoresist, silicon surfaces, and ion milled glass substrates are presented. Primary rat calvarial osteoblasts were grown on ion-milled glass substrates with nanotopography with a periodicity of 1200 nm. Fluorescent microscopy revealed that cells formed adhesions sites coincident with the nanotopography after 24 h of growth on the substrates. The results suggest that laser LIL is an easy and inexpensive method to fabricate systematically changing nanostructures for cell adhesion studies. The effect of the different periodicities and transition structures can be studied on a single substrate to reduce the number of samples significantly.