Neonatal blood spot testing is offered to all babies in the first week of life as part of the UK Newborn Screening Programme. A blood sample is taken from the heel and used to screen for phenylketonuria, congenital hypothyroidism, cystic fibrosis, sickle cell diseases and medium-chain acyl-CoA dehydrogenase deficiency. This article aims to discuss the principles of screening as applied to neonatal blood spot testing, the blood spot test and the conditions which screening aims to detect.