Background The occlusive patch test developed for assessing topical retinoids activity in human skin has been extended as a short-term screening protocol for anti-ageing agents. In this model, biopsies are performed at the end of the occlusion period for morphological and immuno-histochemistry analysis. Multiphoton microscopy is a recent non-invasive imaging technique that combined with image processing tools allows the in vivo quantification of human skin modifications. Objective To validate with gold standards of anti-ageing that are retinoids, the relevance of multiphoton microscopy for kinetic and quantitative assessment in this model. Methods Twenty women, aged 50–65 years, were enrolled. Retinol 0.3% (RO) and Retinoic acid 0.025% (RA) were applied to the dorsal photo-damaged side of their forearm under occlusive patches for 12 days. A patch alone was applied to a third area as control. Evaluation was performed at day D0, D12 (end of treatment), D18 and D32 using multiphoton microscopy. Epidermal thickness, normalized area of the dermal-epidermal junction (DEJ) and melanin density were estimated using 3D image processing tools. Results Main significant results are: Epidermal thickening at D12, D18 and D32 with RO and at D12, D18 with RA vs. baseline and vs. control. Increased DEJ undulation at D32 with RO and at D12 with RA vs. baseline and vs. control. Decreased melanin content with RO (at D12 and D18 vs. baseline and at D32 vs. baseline and vs. control) and with RA (at D12 vs. baseline). Conclusions This study shows that multiphoton microscopy associated to specific 3D image processing tools allows cutaneous effects induced by topical retinoids in this in vivo model to be non-invasively detected, quantified and followed over time. This innovative approach could be applied to the evaluation of other active compounds.