This study describes the optimized stages of lipid extraction assisted by ultrasound to increase the concentrations of limonoids and steroids from andiroba seeds, identified as Carapa guianensis Aublet, and the lipidome analyzed by TLC and GC/MS. After boiling and peeling, crushed seeds were subjected to extractions with n-hexane (G1), acetone (G2), and methanol (G3) in an ultrasonic bath for 30 minutes at 50 °C. These extracts were analyzed by analytical TLC; aliquots of each extract and a marketable oil, used as a reference, were transesterified followed by silylation with BSTFA + 1% TMCS, and analyzed by GC/MS. The reference oil and the biomass of the seeds did not present significant differences in the profiles of free fatty acids (FFA). However, differences were observed in the profiles of tetranortriterpenoids (TnTT) (limonoids) in the seeds. Afterward, the analysis of the extracts G1, G2, and G3 detected free steroids: campesterol, stigmasterol, and β-sitosterol; and limonoids: 7-deacetyl-7-oxogedunin, 6α-acetoxy-gedunin, deacetylgedunin, and epoxyacetylgedunin. The use of lipidomic techniques associated with ultrasound-assisted extraction was applied for the first time to enhance the triterpenoids and steroids, mainly in G3, describing a faster and more economical process, and allowing a one-step lipidome analysis of the andiroba seeds.