Assessment of craniofacial skeletal maturity is of great importance in orthodontic diagnosis and treatment planning. Traditional radiographic methods suffer from clinician subjectivity and low reproducibility. Recent biochemical methods, such as the use of gingival crevicular fluid (GCF) protein biomarkers involved in bone metabolism, have provided new opportunities to assess skeletal maturity. However, mass spectrometry (MS)-based GCF proteomic analysis still faces significant challenges, including the interference of high abundance proteins, laborious sample prefractionation and relatively limited coverage of GCF proteome. To improve GCF sample processing and further discover novel biomarkers, we herein developed a single-pot, solid-phase-enhanced sample-preparation (SP3)-based high-field asymmetric waveform ion mobility spectrometry (FAIMS)-MS protocol for deep quantitative analysis of the GCF proteome for skeletal maturity indicators. SP3 combined with FAIMS could minimize sample loss and eliminate tedious and time-consuming offline fractionation, thereby simplifying GCF sample preparation and improving analytical coverage and reproducibility of the GCF proteome. A total of 5407 proteins were identified in GCF samples from prepubertal and circumpubertal groups, representing the largest dataset of human GCF proteome to date. Compared to the prepubertal group, 61 proteins were differentially expressed (31 up-regulated, 30 down-regulated) in the circumpubertal group. The six-protein marker panel, including ATP5D, CLTA, CLTB, DNM2, HSPA8 and NCK1, showed great potential to predict the circumpubertal stage (ROC-AUC 0.937), which provided new insights into skeletal maturity assessment. Display omitted •SP3 combined with FAIMS simplified GCF sample processing, eliminating the common but laborious sample fractionation.•The SP3-based FAIMS-MS protocol improved the coverage of GCF proteome and showed good quantitative performance (R2 > 0.98).•We generated the largest dataset of human GCF proteome (5407 proteins) to date.•A novel six-protein marker panel (AUC = 0.937) was provided for skeletal maturity assessment.