Dissecting temporally the sequence of secondary structural changes, and determining how these specific changes modulate conformational heterogeneity, remain major goals of protein folding studies. In this study, the folding of the SH3 domain of PI3 kinase has been characterized using pulsed hydrogen exchange mass spectrometry (HX-MS). The folding could be described as a four-state process, U ↔ IVE ↔ IE ↔ N, where IVE and IE are structurally heterogeneous intermediate ensembles. Compared to U, early intermediate IVE has a marginally increased level of protection against HX of amides along the entire length of the polypeptide. Sequential assembly into β-sheet structure has been resolved temporally. Three of the five β-strands acquire nativelike structure before the rate-limiting step. β-Strands 2 and 5 acquire nativelike structure in IVE, while β-strand 4 does so in IE. β-Strand 1 acquires nativelike structure only during the last step of the folding process. Hence, the HX-MS study has resolved the order of assembly of the β-strands for the formation of the two β-sheets, which previous studies utilizing Φ-value analysis of several different SH3 domains had been unable to accomplish. Moreover, it is shown that structural heterogeneity decreases in a stepwise manner during the three stages of folding.