Photodynamic therapy (PDT) with methyl-aminolevulinate (MAL) is anapproved non-invasive treatment option for actinic keratoses (AKs). In vivo reflectanceconfocal microscopy (RCM) is a non-invasive tool for real-time imaging of epidermis andsuperficial dermis in vivo that has been previously reported to facilitate the in vivoevaluation of skin lesions, including AKs. The aim of this study was to investigate the useof in vivo RCM in evaluating AKs response to MAL-PDT. For this reason a total of10 biopsy-proven AKs were treated by MAL-PDT, according to standard PDT protocol forAKs. RCM investigation was performed before and after PDT and RCM-guided punchbiopsies was taken at 3 months in all patients for histopathologic examination. At 3 monthsfollow-up, complete clinical response was observed by clinical examination in 9 out of10 lesions and a partial clinical response in 1 lesion. In vivo RCM detected two residualAKs in subclinical form, missed by clinical examination. Histological analysis confirmedthese results. In vivo RCM may be a new alternative tool for the non-invasive diagnosis ofAKs and evaluation of AKs response to non-invasive treatments, as MAL-PDT, improvingthe ability of dermatologists to diagnose AKs even in subclinical stage.