Using laser excitation, expression microdissection (xMD) can selectively heat cancer cells targeted via immunohistochemical staining to enable their selective retrieval from tumor tissue samples, thus reducing misdiagnoses caused by contamination of noncancerous cells. Several theoretical models have been validated for the photothermal effect in highly light absorbing and scattering media. However, these models are not generally applicable to the physics behind the process of xMD. In this study, we propose a thermal model that can analyze the transient temperature distribution and heat melt zone in an xMD sample medium composed of a thermoplastic film and a tumor tissue sample sandwiched between two glass slides. Furthermore, we experimentally examined the model using an ink layer with controllable optical properties to serve as a microscale-thin, tissue-mimicking phantom and found the experimentally measured film temperature is in good agreement with the model predictions. The validated model can help researchers to optimize cell retrieval by xMD for improved diagnostics of cancer and other diseases.