A sex-gender approach in laboratory medicine is scarce; furthermore, the influence of sex on acylcarnitines and amino acid levels at birth has not been thoroughly investigated, even if sex impacts on newborn screening. We aimed to establish the influence of sex on amino acids and acylcarnitines levels in male and female newborns. Amino acids and acylcarnitines were analysed in dried blood spots using tandem mass spectrometry in male and female newborns. Data were analysed before and after body weight correction also using principal components analysis. This retrospective analytical study showed that females had small but significantly higher levels of amino acids and the correction for body weight amplified these differences. Acylcarnitines were overall higher in males before body weight correction with the exception of isovalerylcarnitine + methylbutyrylcarnitine (C5), which was significantly higher in females. Body weight correction decreased the sex differences in C5. Principal component analysis showed that both amino acids and acylcarnitines were necessary to describe the model for females, whereas only acylcarnitines were required for males. These metabolomics data underline the importance of including sex as a variable in future investigations of circulating metabolites; the existence of sex differences highlights the need for setting distinct reference values for female and male neonates in metabolite concentration.