DEER spectroscopy applied to orthogonally spin-labeled proteins is a versatile technique which allows simplifying the assignment of distances in complex spin systems and thereby increasing the information content that can be obtained per sample. In fact, orthogonal spin labels can be independently addressed in DEER experiments due to spectroscopically non-overlapping central transitions, distinct relaxation times and/or transition moments. Here we focus on molecular rulers orthogonally labeled with nitroxide (NO) and gadolinium (Gd) spins, which give access to three distinct DEER channels, probing NO-NO, NO-Gd and Gd-Gd distances. It has been previously suggested that crosstalk signals between individual DEER channels might occur, for example, between NO and Gd due to their inevitable spectral overlap. However, a systematic study to address these issues has not yet been carried out. Here, we perform a thorough three-channel DEER analysis on mixtures of NO-NO, NO-Gd and Gd-Gd molecular rulers characterized by distinct, non-overlapping distance distributions to study under which conditions crosstalk signals occur and how they can be identified or suppressed to improve signal fidelity. This study will help to improve the assignment of the correct distances in homo- and hetero-complexes of orthogonally spin-labeled proteins.