Objective: Lipids are secreted into milk as bilayer-coated structures: milk lipid globules (MLGs). Adipophilin (ADRP) and perilipin 3 (TIP47) are associated with MLGs in human breast milk; however, the role of these proteins in milk lipid secretion is not fully understood. The aim of the study was to investigate levels of ADRP, TIP47 and total lipid content in human breast milk, their mutual correlations and dynamics during lactation. Research Methods & Procedures: Milk samples from 22 healthy lactating women (Caucasian, Central European) were collected at five time points during lactation (1–3, 12–14, 29–30, 88–90 and 178–180 days postpartum). Mass spectrometry-based method was used for quantification of ADRP and TIP47 in the samples. The gravimetric method was used to determine milk total lipid content. Results: We observed distinctive trends in ADRP, TIP47 levels and lipid content in human breast milk during the first 6 months of lactation. We also found a significant association between lipid content and ADRP, lipid content and TIP47, and ADRP and TIP47 concentrations in breast milk at all sampling points. Moreover, we derived an equation for estimating the mean lipid content of milk based on ADRP concentration in human breast milk. Conclusions: A mass spectrometry-based method was developed for quantifying ADRP and TIP47 in human breast milk. Strong mutual correlations were found between ADRP, TIP47 and total lipid content in human breast milk.