The regulation and mechanism of outer membrane (OM) lysis during spore wall assembly is not well understood. Although the timing of OM lysis has been established in previous studies, this event has never been assayed for. To this end, a new method was developed to assay the lysis of the OM during sporulation in Saccharomyces cerevisiae, the split GFP assay. By expressing complementary fragments of GFP in the ascal cytoplasm and the lumen between the OM and the spore plasma membrane (SPM), the lysis of the OM could be detected by the reconstitution of a functional GFP protein in the ascal cytoplasm as a result of these two compartments merging. A screen of several mutant strains with sporulation defects with published TEM images showing whether or not the lyse the OM then verified the split GFP assay for OM lysis as an effective method for detecting this event. This assay was then used to further investigate the sporulation defects associated with mutations in FKS genes, which all have 1,3-β-glucan synthase homology. The results of this assay showed that mutation in FKS2 or FKS3 leads to a reduction on observed OM lysis. This provides further evidence for the current model that proper completion of the β-glucan layer needs to occur before the OM will lyse. In addition, it was shown via dissection that FKS3 is not able to substitute for FKS1, as FKS2 has been shown to do. Although FKS3 is very similar to the other 1,3-β-glucan synthases homologically, the data suggest it has a function distinct from that of FKS1 and FKS2.