Autoregulation capacity has been classically assessed with a ‘two-point’ measurement or static autoregulation (sAR). In such an approach, stabilized hemodynamic parameters are determined before and after a perfusion pressure challenge. Analysis of dynamic autoregulation (dAR), an early phase of blood flow response to a sudden perfusion pressure change is emerging as a preferred approach to assess the capacity of autoregulation in many non-ocular tissues and has developed rapidly in the last decade. The purpose of this study was to develop a method to quantify dAR in the optic nerve head (ONH). In six pentobarbital (6–9 mg/kg/h, IV) anesthetized rhesus monkeys, dAR was elicited by increasing intraocular pressure (IOP) from 10 to 30 or 40 mmHg (IOP 10–30 /IOP 10–40 ) manometrically via switch between reservoirs connected to the anterior chamber. Relative blood flow changes during dAR in the ONH, estimated with a laser speckle flowgraph (LSFG), were continuously measured for 1 min. Time-domain parameters of dAR response, including: BF Δmax (maximal blood flow decrease, %), K r (descending slope of blood flow from baseline to BF Δmax ) and T r (descending time of blood flow from baseline to BF Δmax ) were extracted and analyzed offline. For each monkey, same procedure was repeated three times during three different visits. The test-retest repeatability and inter-ocular difference of the parameters was statistically evaluated. During IOP 10–30 and IOP 10–40 , the mean arterial BP was 89 ± 7 and 85 ± 6 mmHg, respectively. Immediately after the reservoir was switched, the blood flow started to decline and reached maximal in ∼4 s. The blood flow then returned back toward baseline despite continuous IOP increase, which took 8–11 s to reach the level of the raised reservoir. The general pattern of blood flow responses was similar between IOP 10–30 and IOP 10–40 and there was no statistically significant difference for T r (P > 0.05). However, IOP 10–40 caused greater BF Δmax and deeper K r than IOP 10–30 (P < 0.0001 and P < 0.05, respectively). The blood flow during steady state, 5 min after IOP elevation, showed no statistically significant difference from baseline (P > 0.05). All dAR parameters (T r , K r and BF Δmax ) showed no significant difference across the 3 visits (Repeat measures ANOVA, P = 0.7, 0.2 and 0.2, respectively); the corresponding coefficients of variance were 24%, 43% and 34% during IOP 10–30 and 11.8%, 30.3% and 19.0% during IOP 10–40 . The mean dAR parameters between the eyes showed no statistically differences (P = 0.6) during both IOP 10–30 and IOP 10–40 . The current study showed that a rapid ocular perfusion pressure decrease induced by a sudden IOP step increase evoked a transient and reproducible dAR response in the ONH of non-human primates measured with LSFG. Quantitative analysis of dAR may provide a direct view of vasomotorial activity in the resistant vessels and thus a new approach to assess the autoregulatory capacity in the ONH.