The antineoplastic drug hydroxyurea (HU), when used at subtoxic doses, induces prolonged replication stress and centrosome amplification. This causes genomic instability and increases the malignancy of the recurring tumor. The mechanism of centrosome amplification induced by prolonged replication stress, however, is still unclear. Here, we examined the involvement of ataxia telangiectasia, mutated (ATM), ataxia telangiectasia, mutated and Rad3-related (ATR) and DNA-dependent protein kinase (DNA-PK) and found that HU-induced centrosome amplification was inhibited by the depletion of DNA-PKcs, but not ATM and ATR. Inactivation of ATM/ATR in U2OS cells instead caused aneuploidy and cell death. We found DNA-PKcs depletion also abrogated ATM phosphorylation, indicating that ATM activation during prolonged replication stress depends on DNA-PK. Depletion of DNA-PK abrogated checkpoint kinase (Chk)2 activation and partially reduced Chk1 activation. Chk2 depletion blocked HU-induced centrosome amplification, indicating a function of Chk2 in centrosome amplification. We further found that Chk2 was phosphorylated at Thr68 on the mother centriole at late G2 and mitosis when unstressed and on all amplified centrioles induced by HU. In summary, we have elucidated that DNA-PK/Chk2 signaling induces centrosome amplification upon long-term HU treatment, therefore increasing our insight into tumor recurrence after initial chemotherapy.