Three-dimensional microstructured scaffolds provide a means for cells to be cultured in vitro in a way that resembles natural conditions more closely than flat tissue culture polystyrene. In the presented work, two-photon polymerization (2PP) is applied as a tool for the engineering of high-resolution 3D scaffold structures with a well defined microarchitecture made of biocompatible photo resins. 2PP is a novel photolithographic technique using femtosecond laser pulses which enables free 3D microstructuring of liquid photo resins due to the relationship of the axial and lateral spatial confinement of the photoreaction to the focal volume of a focused laser beam. A set of photo resins were tested with regard to 2PP processability and three different classes of methacrylated photopolymerizable monomers (methacrylated oligolactones, urethane dimethacrylate, poly(ethylene glycol diacrylate)) were found to be efficient 2PP materials. 3D microstructures based on computer models were produced and tested for biocompatibility. The initial cell adhesion and the viability of bovine chondrocytes on the polymeric scaffolds were evaluated morphologically by confocal laser scanning microscopy (CLSM) after three-day culture on 2PP derived microstructures. 2PP derived scaffolds were fabricated in different sizes and geometries, starting from the 100 µm-range reaching out to the cm-range showing the actual possibilities to produce large volume scaffolds even for implantation purposes.