The search for an islet β-cell growth factor has been a key objective in recent diabetes research, since the ability to regenerate and/or protect the functioning β-cell population in patients could result in a great advancement for diabetes treatment. Insulin-like growth factor (IGF)-I and IGF-II are known to play crucial roles in fetal growth and prenatal development, and there is growing evidence that IGF-II increases β-cell proliferation and survival in vitro and in vivo. A search for the source of IGF-II-like-immunoreactivity in isolated β-cell secretory granules from the murine cell line βTC6-F7 revealed a novel two-chain IGF-II-derived peptide which we named vesiculin and which has been shown to be a full insulin agonist. Here, we present an LC-MS/MS method that enables selective detection and semi-quantitation of the highly related IGF-II and vesiculin molecules. We have used this method to measure these two peptides in conditioned media from two β-cell lines, produced under increasing glucose concentrations. This technique detected both IGF-II and vesiculin in media conditioned by MIN6 and βTC6-F7 cells at levels in the range of 0–6 μM (total insulin 80–450 μM), and revealed a glucose-stimulated increase in insulin, IGF-II and vesiculin. IGF-II was detected in adult human and neonatal mouse serum in high levels, but vesiculin was not present. The methodology we present herein has utility for detecting and differentiating active peptides that are highly related and of low abundance.