Currently there is a paucity of inhibitors that can be used as molecular probes to inhibit cell cycle enzymes in an acute and temporal manner, which is critical to understanding their functions. Additionally, these compounds could be developed into therapies to treat proliferative diseases like cancer. We report the identification of novel chemical probes for dissecting the mechanisms governing cell cycle progression with an emphasis on cell division and for developing novel anticancer therapeutics. We devised a cancer cell-based high-throughput chemical screening approach to identify cell cycle modulators. Briefly, a cell cycle profile was generated for each drug to measure the cellular response to each drug. We identified G1, S, G2, and M-phase specific inhibitors. To determine their intracellular targets, we developed CSNAP (Chemical Similarity Network Analysis Pulldown), a computational prorgam that searched the ChEMBL database for compounds sharing chemical similarity to hit compounds, retrieved the bioactivity information (including targets), organized these compounds into network similarity graphs and inferred targets based on the frequency of compound targets in the neighborhood of query compounds. This revealed analogs of known G1, S, G2, and M inhibitors and new compounds with unknown targets, indicating that our screening discovered validated and novel structurally diverse cell cycle phase-specific inhibitors. We further performed a multiparametric pehnotypic analysis of each mitotic inhibitor and analyzed their potency. We highlight novel inhibitors of mitotic spindle assembly and their mechanism of action and debut the open access CSNAP web server for the scientific community.