The presence and integrity of amniotic fluid is fundamental for the normal development of the human fetus during pregnancy. Its production rate changes throughout pregnancy and is mainly related to the functions of the different fetal, placental and amniotic compartments. Premature rupture of the membranes (PROM) occurs in about 5% of deliveries, with complications such as infection and preterm birth. The management of patients with PROM, regardless of gestational age, remains controversial, and it is therefore important to develop new biological tests in order to achieve accurate diagnoses by identifying the presence of specific amniotic fluid markers in vaginal environment. We recently showed the usefulness of amniotic fluid proteomics in identifying a series of peptides that were absent from the corresponding maternal plasma. Several peptides corresponded to fragments of plasma proteins. Two peptides, absent from plasma samples of pregnant women, were identified in amniotic fluid. They corresponded to the COOH-terminal parts of perlecan (SwissProt: P98160) and of agrin (SwissProt: O00468) protein cores, two major heparan sulfate proteoglycans of basement membranes. In this review we will discuss modern proteomic strategies that may improve the laboratory assessment of PROM, and will focus on some of the biochemical characteristics of agrin and perlecan fragments identified in amniotic fluid.