Clayton was born in Liverpool. She attended Bromley County School for Girls (London), where she was head girl. She went on to study medicine at Edinburgh University, qualifying in 1946. She met William Klyne in 1947 while both were employed at the Medical Research Council; they married in 1949, and remained married until William Klyne's death. The couple had two children.
The couple moved from Edinburgh to London, where Clayton began working in the chemical pathology department at St Thomas's Medical College, where, using new biochemistry techniques, her research on hormones brought her widespread recognition. 
- 1988: For her contributions to science she was named a Dame Commander of the Order of the British Empire in 1988.
- 1969: she was awarded a PhD for her research on oestrogens.
- Barbara Evelyn Clayton, at the Lancet, Volume 377, Issue 9775, p. 1402, 23 April 2011(doi:10.1016/S0140-6736(11)60564-3); by Caroline Richmond; retrieved 29 May 2013.
- Profile in The Guardian; accessed 26 March 2014.
- Profile in Who's Who, ukwhoswho.com; accessed 26 March 2014.
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