Louisiana pine snakes (Pituophis ruthveni) are a threatened species with a range limited to portions of Louisiana and Texas. There are captive reproduction programs that aim to assist in the recovery of this species. Therefore, obtaining information that can aid in maintaining their health, such as a complete ocular examination, is a critical component in conservation efforts. Examination of the snake ocular posterior segment is challenging because of the presence of striated uveal musculature that is unresponsive to anticholinergic agents, and the presence of the spectacle, which may create a barrier to topical mydriatics. The objectives of this study were to document normal ocular biometric measurements (axial globe length [AP], lens thickness (LT), and vitreous chamber depth [VCD]) in this species using ultrasonography (US) and to assess the effects of topically applied rocuronium bromide (RB) solution (one- and two-drop protocol, 20 µl and 40 µl, respectively) to facilitate noninvasive examination of the fundus. The ocular biometric measurements were then correlated to morphometric data including snout–vent length (SVL), tail length (TL), total body length (SVL + TL), sex, and body condition score (BCS). We found that US, using an 8–18-MHz linear array probe, provided adequate image quality to measure the above parameters in all snakes (n = 8), and that there were no significant correlations between the morphometric data and ocular biometric US measurements (P > 0.05). RB solution applied topically to the healthy, intact spectacle did not induce mydriasis in a subset of this same population (n = 6; P > 0.05) using either the one- or two-drop protocol. In conclusion, ocular US can be utilized as a noninvasive diagnostic tool in Louisiana pine snakes and further studies are needed to evaluate noninvasive methods of inducing mydriasis in this species.