We have developed a new quantitative native PAGE mobility shift assay, which allows for the measurement of binding affinities for interacting protein pairs, one of which is fluorescently labelled. We have used it to examine recognition of the Simian virus 40 (SV40) large tumour T-antigen (T-ag) nuclear localisation sequence (NLS) by members of the importin (Imp) superfamily of nuclear transport proteins. We demonstrate that the T-ag NLS binds to the Imp alpha/beta heterodimer in NLS-dependent manner, determining that it binds with eight-fold higher affinity (340 nM), when compared to Imp alpha alone, consistent with autoinhibition of Imp alpha when not complexed with Imp beta. The mobility shift assay is able to detect nM binding affinities, making it a sensitive and useful tool to analyse protein-protein interactions in solution.