Mast cells (MCs) are multifunctional cells of the immune system and are found in skin and all major tissues of the body. They contribute to the pathology of several diseases including urticaria, psoriasis, atopic dermatitis and mastocytosis where they are increased at lesional sites. Histomorphometric analysis of skin biopsies serves as a routine method for the assessment of MC numbers and their activation status, which comes with major limitations. As of now, non-invasive techniques to study MCs in vivo are not available. Here, we describe a label-free imaging technique to visualize MCs and their activation status in the human papillary dermis in vivo. This technique uses two-photon excited fluorescence lifetime imaging (TPE-FLIM) signatures, which are different for MCs and other dermal components. TPE-FLIM allows for the visualization and quantification of dermal MCs in healthy subjects and patients with skin diseases. Moreover, TPE-FLIM can differentiate between two MC populations in the papillary dermis in vivo-resting and activated MCs with a sensitivity of 0.81 and 0.87 and a specificity of 0.85 and 0.84, respectively. Results obtained on healthy volunteers and allergy and mastocytosis patients indicate the existence of other MC subpopulations within known resting and activated MC populations. The developed method may become an important tool for non-invasive in vivo diagnostics and therapy control in dermatology and immunology, which will help to better understand pathomechanisms involving MC accumulation, activation and degranulation and to characterize the effects of therapies that target MCs.