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Actinic keratosis (AK) is a common precancerous condition found on sun-damaged skin. Tirbanibulin 1 % ointment has been approved for the topical treatment of non-hyperkeratotic facial and scalp Olsen grade I AKs over a contiguous area of 25 cm2 with a daily application for 5 consecutive days. Our aim was to investigate the use of in vivo RCM in the assessment of the response of AKs treated with tirbanibulin, as it has never been described in the published Literature. A total of 10 AKs in 10 consecutive outpatients were enrolled in the present study in May 2023. The follow-up visit was scheduled after 30 days from last application of tirbanibulin ointment. At follow-up visit, a complete response was described by clinical, dermoscopic and in vivo RCM examination in 10 out of 10 lesions, with a recovery of stratum corneum, decrease in atypical honeycomb pattern and changes in dermal collagen. All patients were followed up for at least 8 months and further recurrences were not registered. Based on our experience, we confirm the efficacy and safety of tirbanibulin in treating AKs and the usefulness of RCM in vivo examination for the therapeutic monitoring of such lesions, even in a very early stage.