The history of chemical probes can be divided into two phases. The first NIH-centric initiative, from 2004 to 2014, is described in the year-six reports of the online book series 'Probe Reports from the NIH Molecular Libraries Program' (NBK47352). It was instigated as the Molecular Libraries Probe Production Centres Network (MLPCN), a US-wide consortium of centres that were to produce probes. They solicited novel assays from the research community for high throughput assays (HTS) against what was initially a library of 350,000 compounds maintained as the Molecular Libraries Small Molecule Repository, MLSMR (this has expanded modestly over the last five years to 406,000). Most primary screens went through an orthogonal second round designated as confirmatory assays in PubChem BioAssay. Validated hits were then optimised by medicinal chemistry (usually from PIs collaborating with the centres and sometimes filling patents) to produce in vitro chemical probes with structures and assay results eventually deposited into PubChem (indeed, this was the raison d'être for setting up PubChem in 2004).