The decrease in firmness is a hallmark of skin aging. Accelerated by chronic sun exposure, fundamental changes occur within the dermal extracellular matrix over the years, mainly impairing the collagenous network. Based on the qualitative and quantitative assessment of skin firmness, in vitro and in vivo studies were carried out to elucidate the effects of topical folic acid and creatine to counteract this age-dependent reduction in the amount of collagen. Topical application of a commercially available formulation containing folic acid and creatine was performed to study effects on skin firmness in vivo using cutometric analysis. Imaging and quantification of collagen density were carried out using multiphoton laser scanning microscopy (MPLSM). To investigate the effects of these compounds on collagen gene expression, procollagen synthesis, and collagen fibril organization, complementary in vitro studies on cultured fibroblast-populated collagen gels were carried out. The underlying structural changes in the collagen network of young and aged sun-exposed facial skin in vivo were visualized by MPLSM. Topical application of a folic acid- and creatine-containing formulation significantly improved firmness of mature skin in vivo. Treatment of fibroblast-populated dermal equivalents with folic acid and creatine increased collagen gene expression and procollagen levels and improved collagen fiber density, suggesting that the in vivo effects are based on the overall improvement of the collagen metabolism. Employing MPLSM, dermal changes occurring in photo-aged human skin were visualized in an unprecedented manner and correlated to a loss of firmness. Treatment of aged skin with a topical formulation containing folic acid and creatine counteracted this age-dependent decline by exerting sustained effects on collagen metabolism. Our results support previous findings on the efficacy of these actives.