Background: Non-small cell lung cancer (NSCLC) is the most commonly diagnosed cancer, and radiotherapy (RT) is used for the cancer therapy. RT affects DNA and causes DNA double-strand breaks which are repaired by DNA repair protein ataxia telangiectasia mutated (ATM). RT also affects the mitochondria which is a key player in mediating the radiation response in tumors and removing damaged mitochondria through mitophagy. During mitophagy, Parkin accumulates on defective mitochondria to mediate the clearance of damaged mitochondria. This study examines the effect of radiation on mitophagy using Parkin and ATM antibodies on the human NSCLC A549 line. Materials and Methods: A549 cells were treated with 2, 4, 6 and 8 Gy of radiation were analyzed on days 1 and 3 after a single dose of radiotherapy. Parkin and ATM expressions of A549 cells were examined by using immunohistochemical technique. Results: In the control groups, weak immunoreactivity of ATM and Parkin was observed on both days 1 and 3. The most intense ATM expression was seen in the 6 and 8 Gy groups after day 1. The most intense Parkin expression was seen after the days 1 and 3 in the 2 Gy groups. Parkin immunoreactivity decreased due to increasing radiation dose. Conclusion: It must be considered that mitophagy mechanisms are activated in RT applications. It must be considered that the activation of mitophagy mechanisms in RT and A549 lung cancer cell lines may provide hemostasis in cancer cells. Molecules targeting mitophagy must be developed for use with radiotherapy.