PURPOSE: To examine big-bubble (BB) formation success rates in deep anterior lamellar keratoplasty (DALK) at various corneal depths using real-time guidance from swept-source, microscope-integrated optical coherence tomography (SS-MIOCT). METHODS: The DALK procedure was performed ex vivo with 34 human donor corneoscleral buttons on pressurized artificial anterior chambers using the BB technique employed by Anwar and Teichmann. We inserted a needle under controlled ex vivo conditions to corneal depths ranging from 40% to ≥90% using real-time guidance from SS-MIOCT and injected air. BB success was then determined for each injection. RESULTS: The average needle depth for successful full BB formation was 79.9% ± 3.0% compared with 66.9% ± 2.6% for partial BB formation and 49.9% ± 3.4% for no BB formation (P < 0.0001). Expressed as stroma below the needle tip, this corresponded to 123.9 ± 20.0 μm for successful full BB formation compared with 233.7 ± 23.8 μm for partial BB formation and 316.7 ± 17.3 μm for no BB formation (P < 0.0001). All other variables tested (sex, race, age, endothelial cell density, air injected, needle angle, and central corneal thickness) did not significantly affect BB formation success rates. CONCLUSIONS: BB formation in DALK is more successful if needle insertion and air injection occur at deeper corneal depth. However, ≥90% corneal depth was not necessary in this ex vivo model of DALK. SS-MIOCT can be used to accurately guide the needle in real time.