High-throughput screening (HTS) is an effective method for lead and probe discovery that is widely used in industry and academia to identify novel chemical matter and to initiate the drug discovery process. However, HTS can be time consuming and costly and the use of subsets as an efficient alternative to screening entire compound collections has been investigated. Subsets may be selected on the basis of chemical diversity, molecular properties, biological activity diversity or biological target focus. Previously, we described a novel form of subset screening: plate-based diversity subset (PBDS) screening, in which the screening subset is constructed by plate selection (rather than individual compound cherry-picking), using algorithms that select for compound quality and chemical diversity on a plate basis. In this paper, we describe a second-generation approach to the construction of an updated subset: PBDS2, using both plate and individual compound selection, that has an improved coverage of the chemical space of the screening file, whilst only selecting the same number of plates for screening. We describe the validation of PBDS2 and its successful use in hit and lead discovery. PBDS2 screening became the default mode of singleton (one compound per well) HTS for lead discovery in Pfizer.