Many questions in cell biology and biophysics involve the quantitation of the colocalisation of proteins tagged with different fluorophores and their interaction. However, the incomplete separation of the different colour channels due to the presence of autofluorescence, along with cross-excitation and emission ‘bleed-through’ of one colour channel into the other, all combine to render the interpretation of multiband images ambiguous. Traditionally often used in a qualitative manner by simply overlaying fluorescence images (‘red plus green equals yellow’), multicolour fluorescence is increasingly moving away from static dual-colour images towards more quantitative studies involving the investigation of dynamical three-dimensional interaction of proteins tagged with different fluorophores in live cells. Quantifying fluorescence resonance energy transfer efficiency, fluorescence complementation and colour merging following photoactivation or photoswitching provide related examples in which quantitative image analysis of multicolour fluorescence is required. Despite its widespread use, reliable standards for evaluating the degree of spectral overlap in multicolour fluorescence and calculating quantitative colocalisation estimates are missing. In this chapter, using a number of intuitive yet practical examples, we discuss the factors that affect image quality and study their impact on the measured degree of colocalisation. We equally compare different pixel-based and object-based descriptors for analysing colocalisation of spectrally separate fluorescence. Finally, we discuss the use of spectral imaging and linear unmixing to study the presence in a ‘mixed pixel’ of spectrally overlapping fluorophores and discuss how this technique can be used to provide quantitative colocalisation information in more complex experimental scenarios in which classic dual- or triple-colour fluorescence would produce erroneous results.