Application of ultrasound (US) to evaluate attainment and morphology of glandular tissue provides a new rationale for evaluating onset and progression of female puberty, but currently no hormone references complement this method. Further, previous studies have not explored the predictive value of endocrine profiling to determine female puberty onset.To integrate US breast staging with hypothalamic-pituitary-gonadal hormone references and test the predictive value of an endocrine profile to determine thelarche.Cross-sectional sample of 601 healthy Norwegian girls, age 6 to 16 years.Clinical and ultrasound breast evaluations were performed for all included girls. Blood samples were analyzed by immunoassay and ultrasensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify estradiol (E2) and estrone (E1) from the sub-picomolar range.References for E2, E1, LH, FSH and SHBG were constructed in relation to chronological age, Tanner and US breast stages. An endocrine profile index score derived from principle component analysis of these analytes was a better marker of puberty onset than age or any individual hormone, with ROC AUC 0.91 (P<0.001). Ultrasound detection of non-palpable glandular tissue in 14 out of 264 (5.3%) girls with clinically prepubertal presentation was associated with significantly higher median serum levels of E2 (12.5 vs 4.9 pmol/L; P<0.05) and a distinct endocrine profile (arbitrary units; P<0.001).We provide the first hormone references for use with US breast staging and demonstrate the application of endocrine profiling to improve detection of female puberty onset.