Squamous cell carcinoma (SCC) of the skin is a malignant neoplasm that occurs in all ethnic groups primarily due to chronic sun exposure and constitutes a major health problem worldwide. Novel therapies for SCC are in development but as yet no in vitro models capable of screening these therapies and their mechanism of action before proceeding to clinical trials in human subjects have emerged. For this reason we have developed and characterized a novel three-dimensional human SCC construct and validated it using photodynamic therapy (PDT), a well-established modality for treating in situ SCCs. Histologic and immunohistochemical characterization of these SCC constructs revealed epidermal and dermal de-differentiation, increased cell proliferation and expression of immunohistochemical markers specific for cutaneous SCC. Application of PDT to these constructs led to tumor regression with widespread apoptosis and necrosis within 5 days. This in vitro model consistently reproduces the tumor development and dynamics of growing SCCs in vivo and provides a useful approach for screening new treatment modalities for this form of cutaneous cancer.