Tumour spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the spheroid at cell level is important for analysis, yet existing techniques are often expensive, laborious, use non-standard equipment, cause significant size distortion, or are limited to relatively small spheroids. This protocol presents a new method of mounting and clearing spheroids that addresses these issues while allowing for confocal analysis of the inner structure of spheroids. In contrast to existing methods, this protocol allows for rapid mounting and clearing of large numbers of spheroids using standard equipment and laboratory supplies. Mounting spheroids in a pH-neutral agarose-PBS gel solution before introducing a refractive-index-matched clearing solution minimises size distortion common to other similar techniques. This allows for detailed quantitative and statistical analysis where the accuracy of size measurements is paramount. Furthermore, compared to liquid clearing solutions, the agarose gel technique keeps spheroids fixed in place, allowing for the collection of three-dimensional confocal images. We demonstrate how the method yields high-quality two- and three-dimensional images that provide information about inter-cell variability and inner spheroid structure.