The problem of quantitative numerical simulation of electron paramagnetic resonance (EPR) spectra of biradical probes in both isotropic and aligned media was solved for the first time. The models suitable for the description of the spectra of the probes, both in the rigid limit and in the presence of rotational motions, were developed and successfully applied to model systems. The simulation of EPR spectra allows obtaining the following information about the molecular structure and dynamics: the values of orientation order parameters, the type of rotation mobility and its quantitative characteristics, and the sign and value of the spin exchange constant of the biradical. Model systems used in this work include solutions of nitroxide biradicals in a viscous solvent (squalane) in the range of temperatures 100–370 K and in the aligned liquid crystal n-octylcyanobiphenyl (8CB, 100–298.5 K). Unexpectedly, it was found that in 8CB the main orientation axis of the biradical molecule is perpendicular to the longest molecular axis.