Pulsed electron paramagnetic resonance (EPR) methods such as ESEEM, PELDOR, relaxation time measurements, transient EPR, high-field/high-frequency EPR, and pulsed ENDOR, have been used successfully to investigate the local structure and dynamics of paramagnetic centers in biological samples. These methods allow different contributions to the EPR spectra to be distinguished and can help unravel complicated EPR spectra consisting of overlapping resonance lines, as are often found in disordered protein samples. The basic principles, specific potentials, technical requirements, and limitations of these advanced EPR techniques will be reviewed together with recent applications to metal centers, organic radicals, and spin labels in proteins.