The cleaning of painted artworks, i.e. the critical operation whereby materials are selectively removed from a painted surface by partial thinning or complete elimination of varnish, is one of the most debated conservation operations, being an irreversible process, which may result in chromatic and morphological variations in the painted surface. Due to ageing, the upper layer is subject to darkening and yellowing because of blanching and fading from ultraviolet exposure, dust deposition, and overpainted layers due, for instance, to restoration interventions. This degradation can either alter the original appearance of painting polychromy or cause mechanical failure of the finishes. To address these adverse conditions, a process of examination and analysis is critical to the definition and interpretation of the varnish layer. When investigating the ageing process of old paintings, it is of great importance to obtain insight into the painting technique as practiced in the past, and the first step in gaining this knowledge is, to a large extent, based on the study of the varnish film. An effective control of the process and objective evaluation of its outcome requires therefore instrumental/analytical support. The present study illustrates the successful application of non-invasive optical techniques-such as colorimetry, multispectral reflectography, laser scanning micro-profilometry, and optical coherence tomography-to the monitoring of an Italian fourteenth-century painting cleaning process. Results presented here confirm that optical techniques play a pivotal role in artwork diagnostics, especially with regard to conservation operations, while also indicating their validity when applied to the monitoring of the cleaning process.