With captive invertebrates increasing in popularity, a safe and effective anesthesia protocol is required for their veterinary care. This study investigated the safety and efficacy of 5% isoflurane anesthesia and 0.26 ml hemolymph collection in subadult to adult emperor scorpions (Pandinus imperator; n = 14). Each scorpion was placed in a 2-L anesthetic chamber and induced with 5% isoflurane at a flow rate of 2 L/min oxygen. Anesthetic depth was assessed by rolling the animal into dorsal recumbency and rating the response from one to four: 1 = successful coordinated righting movement, 2 = successful uncoordinated righting movement, 3 = unsuccessful righting movement, 4 = no movement. Ratings 3 and 4 were both adequate for the hemolymph collection. The mean induction time was 4.2 min to reach a level 3 depth and 10.5 min to reach a level 4 depth. All animals were clinically normal for at least 1 yr post experiment. A mean hemolymph volume drawn of 1.92% of body weight, and an anesthetic induction with 5% isoflurane, appear safe in subadult to adult emperor scorpions.