Dried blood spots (DBS) have been used in the clinic as a blood sampling technique since the 1960s and have been sampled for small molecules and metabolites by mass spectrometry since the 1990s.Mass spectrometry is now employed in newborn screening for many inborn errors of metabolism,including phenylketonuria, medium-chain acylCoA dehydrogenase deficiency and maple syrup urine disease.Typically, these analyses have been limited to the domain of traditional solution phase electrospray ionisation, but several groups have investigated the potential of ambient ionisation techniques.One area that has been neglected is the analysis of proteins and peptides, beyond haemoglobin, from dried blood spots, despite the fact that dried blood spots are a potentially rich source of protein biomarkers.Here we present an untargeted bottom up proteomics analysis of DBS based on the liquid extraction surface analysis.