Biomarkers in amniotic fluid (AF) include both non-modified and phosphorylated proteins and can be used in the diagnosis of pregnancy-associated pathologic conditions. In this work, an integrated LC-MS method for selective, sensitive and reproducible analysis of phosphorylation in proteins has been applied to AF. Online digestion of (phospho) proteins was coupled with the selective enrichment on a TiO2 trap, and separated by RPLC-MSn of both normal and phosphorylated produced peptides. First, an AF-pooled sample was analyzed and a general map of contained proteins and biomarkers was derived in a single run. Then, individual AF samples were analyzed with a downscaled platform with improved sensitivity. On purpose, a trypsin-based CIMA (R) minidisk was used for online digestion of AF. The obtained protein profile was highly consistent with the one obtained with traditional off-line digestions. Moreover, the use of a specific phospho-enrichment tool followed by LTQ-Orbitrap, enhanced the confidence in the determination of protein phosphorylation state and phosphorylation sites. The phosphorylation sites of IGFBP-1 and osteopontin present in the AF of two individual samples were monitored with a total of 24 and 17 phosphopeptides, respectively, encoding for 12 putative novel phosphorylation sites in addition to known sites.