Diversity-oriented organic synthesis offers the promise of advancing chemical genetics, where small molecules are used to explore biology. While the split-pool synthetic method is theoretically the most effective approach for the production of large collections of small molecules, it has not been widely adopted due to numerous technical and analytical hurdles. We have developed a split-pool synthesis leading to an array of stock solutions of single 1,3-dioxanes. The quantities of compounds are sufficient for hundreds of phenotypic and protein-binding assays. The average concentration of these stock solutions derived from a single synthesis bead was determined to be 5.4 mM in 5 muL of DMSO. A mass spectrometric strategy to identify the structure of molecules from a split-pool synthesis was shown to be highly accurate. Individual members of the 1,3-dioxane library have activity in a variety of phenotypic and protein-binding assays. The procedure developed in this study allows many assays to be performed with compounds derived from individual synthesis beads. The synthetic compounds identified in these assays should serve as useful probes of cellular and organismal processes.