Having developed a reliable and versatile solid-phase strategy for the split-and-pool synthesis of naturally occurring and designed derivatives of the benzopyran template (see preceding paper), we now report the construction of a 10 000-membered natural product-like compound library for chemical biology studies. Concomitantly, we report an early application of the IRORI NanoKan optical encoding system for the high throughput nonchemical tagging and sorting of library members during split-and-pool synthesis. The overall synthetic strategy for library construction is discussed and the individual reaction pathways are examined in the context of specific library members, illustrating reaction conditions as well as yields and purities, The issues of building block selection and quality control of library members are also addressed and, finally, potential applications of the library to chemical biology are discussed.