Polo-like kinase 1 (PLK1), a serine/threonine protein kinase, plays a crucial role in essential biological processes such as cell division, DNA damage response, and cell death. Since its dysregulation is highly associated with tumor development and progression, PLK1 inhibitors, including onvansertib, have been developed as promising anti-cancer therapeutics. Onvansertib is currently under Phase II investigation to evaluate its safety and efficacy in patients with relapsed small cell lung cancer (SCLC). In this study, the efficacy, tolerability, and toxicity of onvansertib were comprehensively profiled using a large panel of 144 cancer cell lines and mouse models. We identified that SCLC cell lines are highly responsive to onvansertib. SCLC xenograft assays showed that daily oral administration of onvansertib at 60 mg/kg was more effective in tumor regression without inducing significant changes in body weight compared to a treatment cycle of 3 days of administration followed by 4 days of rest over a 3-week experimental period. However, in vivo toxicity studies revealed onvansertib-related mortality, clinical signs, and hematological adverse effects at the 60 mg/kg dose. Our findings collectively provide experimental evidence to support combination therapies or toxicity-salvaging low-dose regimens, as reflected in current clinical trials of onvansertib. Additionally, we conducted single-cell RNA-sequencing to elucidate the pharmacological mechanisms of onvansertib in SCLC with unprecedented resolution. Onvansertib impairs a normal cell cycle transition by downregulating the cell division process, leading to G2/M phase arrest. Altogether, this work demonstrates the therapeutic mechanisms and in vitro and in vivo pharmacological profiles of onvansertib in SCLC.