This thesis presents the design, synthesis, and characterization of a 32 Å photoswitchable molecular ruler for DEER (Double Electron-Electron Resonance) spectroscopy. Aimed at addressing the need for reliable and validated distance standards in pulsed dipolar ESR, the ruler features a rigid azobenzene-based spacer that undergoes light-induced cis-trans isomerization, enabling reversible switching from trans to cis conformation and thus giving a second distance. Its narrow distance distribution ensures high-precision measurements and reproducibility across different instruments and analysis methods. The study also establishes a protocol for the preparation of room temperature DEER samples by embedding the ruler in a polystyrene matrix. Overall, this work provides a versatile molecular standard to support more accurate and consistent DEER measurements in both structural biology and materials science.