The eukaryotic cell cycle is a fundamental process in all multicellular organisms and must be precisely regulated to ensure the fidelity of partitioning of the replicated genome to produce two identical daughter cells. Cell cycle corruption is also a hallmark of many cancers and contributes to genomic instability. The replication of the genome (S-phase) is followed by the ‘Gap 2’ or G2 phase then mitosis (M). It is during this time that the sister chomatids are untangled and damaged DNA is repaired. The transition from G2 to mitosis is highly regulated with multiple ‘checkpoints’ that will not be passed if damaged or tangled DNA is detected. One of the proteins involved in the regulation of the G2/M transition is Cyclin B1, which accumulates in the cytoplasm during S- and G2-phases then enters the nucleus at the G2/M transition. Cyclin B1 (encoded by CCNB1) is rapidly degraded at the end of mitosis and is essentially absent during G1-phase. Researchers have exploited this temporal regulation and made a CCNB1-GFP translational fusion reporter construct under the control of native CCNB1 regulatory elements. Here, we describe an siRNA screen of the human kinome in U2OS cells stably transformed with the CCNB1-GFP reporter. This screen was directed at identifying genes required for the progression through G2 into mitosis, such that when those gene products are depleted the cells would arrest in G2 and the Cyclin B1-GFP reporter would accumulate in a large proportion of the cells. The high-content imaging assay used to analyze this screen was developed using an siRNA against CDK1 which encodes Cyclin B1’s binding partner that is required for catalytic activity of the protein kinase complex and initiation of mitosis. This screen identified a small number of genes, some of which are known to be required for the progress of the G2/M transition such as CDK1 and PLK1.