We devised a strategy of 14-3-3 affinity capture and release, isotope differential (d₀/d₄) dimethyl labeling of tryptic digests, and phosphopeptide characterization to identify novel targets of insulin/IGF1/phosphatidylinositol 3-kinase signaling. Notably four known insulin-regulated proteins (PFK-2, PRAS40, AS160, and MYO1C) had high d₀/d₄ values meaning that they were more highly represented among 14-3-3-binding proteins from insulin-stimulated than unstimulated cells. Among novel candidates, insulin receptor substrate 2, the proapoptotic CCDC6, E3 ubiquitin ligase ZNRF2, and signaling adapter SASH1 were confirmed to bind to 14-3-3s in response to IGF1/phosphatidylinositol 3-kinase signaling. Insulin receptor substrate 2, ZNRF2, and SASH1 were also regulated by phorbol ester via p90RSK, whereas CCDC6 and PRAS40 were not. In contrast, the actin-associated protein vasodilator-stimulated phosphoprotein and lipolysis-stimulated lipoprotein receptor, which had low d₀/d₄ scores, bound 14-3-3s irrespective of IGF1 and phorbol ester. Phosphorylated Ser¹⁹ of ZNRF2 (RTRAYpS¹⁹GS), phospho-Ser⁹⁰ of SASH1 (RKRRVpS⁹⁰QD), and phospho- Ser⁴⁹³ of lipolysis-stimulated lipoprotein receptor (RPRARpS⁴⁹³LD) provide one of the 14-3-3-binding sites on each of these proteins. Differential 14-3-3 capture provides a powerful approach to defining downstream regulatory mechanisms for specific signaling pathways.