Metal-free, strain-promoted alkyne–azide cycloaddition (SPAAC) is employed as a versatile technology for the modification of glass with biomolecules. Patterning is executed by stamping of a fluorogenic azidocoumarin or a cyclooctyne to the glass surface, to obtain a unique anchor point for subsequent functionalization by SPAAC. The azidocoumarin at the same time enables straightforward fluorescent read-out of surface reactions. A strong increase in fluorescence is indeed observed upon metal-free reaction with two readily available cyclooctynes, BCN or DIBAC. In addition, functionalized BCN derivatives are employed for glass surface patterning with biotin or even a 27 kDa protein (green fluorescent protein), upon simple incubation.