Carbon monoxide (CO)/nitrogen (N2) separation is a particularly challenging separation, yet it is the one with great industrial relevance for its use in petrochemical synthesis. Although an expensive cryogenic step can be used to perform such separation, it remains ineffective in purifying CO from syngas streams with a significant N2 content. Taking advantage of the lower energy requirement of adsorption processes, we have explored the use of metal–organic frameworks (MOFs) as adsorbents for this difficult separation. We have screened a range of MOF candidates for CO/N2 separation covering a range of chemical and textural features, using the flux response technology to evaluate their dynamic performance for throughput testing alongside equilibrium uptake measurements. We have identified Ni-MOF-74 and Co-MOF-74 as the most promising candidates because of their high metal density and strong metal–CO interactions. We have investigated further the effect of N2 impurity concentrations upon CO/N2 separation using breakthrough adsorption testing and cyclic testing (up to 20 cycles). Overall, using multiple adsorption measurement techniques, this study demonstrates the CO/N2 dynamic separation performance of M-MOF-74 and its ability to be applied for an industrially relevant separation.