Disclosed herein are methods for forming photolithography alignment markers on the back side of a substrate, such as a crystalline silicon substrate used in the manufacture of semiconductor integrated circuits. According to the disclosed techniques, laser radiation is used to remove the material (e.g., silicon) from the back side of a substrate to form the back side alignment markers at specified areas. Such removal can comprise the use of laser ablation or laser-assisted etching. The substrate is placed on a motor-controlled substrate holding mechanism in a laser removal chamber, and the areas are automatically moved underneath the laser radiation to removal the material. The substrate holding mechanism can comprise a standard chuck (in which case use of a protective layer on the front side of the substrate is preferred), or a substrate clamping assembly which suspends the substrate at its edges (in which case the protective layer is not necessary). Alternatively, a stencil having holes corresponding to the shape of the back side alignment markers can be placed over the back side of the substrate to mitigate the need to move the substrate to the areas with precision.