This study compares apoptosis induction in squamous cell carcinoma keratinocytes following exposure to 5-aminolevulinic acid combined with either blue light (417 ± 5 nm) or red light (635 ± 5 nm) photodynamic therapy, with apoptotic activity quantified via levels of Annexin V expression. Our results demonstrate that blue light induces superior levels of apoptosis in squamous cell carcinoma keratinocytes compared to red light at the parameters tested. Both wavelengths demonstrated additional dose- and temperature-dependent effects across two squamous cell carcinoma cell lines. These conclusions have significant implications for the clinical selection of a light source in combination 5-aminolevulinic acid photodynamic therapy in the treatment of actinic keratosis and early squamous cell carcinoma lesions.