Background: In clinical practice, the laryngeal mask airway is an easy-to-use supraglottic airway device. However, the cis-atracurium dosage for laryngeal mask insertion has not been standardised. We aimed to determine the optimal dose of cis-atracurium using a sequential method for successful laryngeal mask insertion. Methods: The cohort study protocol is registered at clinicaltrial.gov (NCT-03668262). Twenty-three patients undergoing elective urinary surgery were sequentially administered cis-atracurium doses as follows: 150, 100, 70, 50, 30, and 20 μg·kg- 1. The main outcome involved the determination of the response to laryngeal mask airway insertion: ≥16 points and < 16 points indicated "satisfactory" and "unsatisfactory" responses, respectively. The median effective dose was estimated using the mean of the seven crossovers from "satisfactory" and "unsatisfactory" responses. The primary outcome involved the determination of the median effective dose (ED50) of cis-atracurium for laryngeal mask airway insertion. Results: The median effective dose of cis-atracurium was 26.5 μg·kg- 1 (95% CI 23.6-29.8) using the sequential method. Heart rate was decreased in the 50 μg·kg- 1 group compared to that in the 30 μg·kg- 1 group at timepoints T7, T8, and T10 (P = 0.0482, P = 0.0460, and P = 0.0236, respectively), but no difference was observed in the 20 μg·kg- 1 group. Systolic blood pressure was decreased in the 50 μg·kg- 1 group compared to that in the 20 μg·kg- 1 group at timepoints T2, T3, and T4 (P = 0.0159, P = 0.0233, and P = 0.0428, respectively). The train-of-four value was significantly lower in the 50 μg·kg- 1 group than in the 30 μg·kg- 1 group at timepoint T3 (P = 0.0326). Conclusions: The ED50 of cis-atracurium was 26.5 μg·kg- 1 for laryngeal mask airway insertion. Trial registration: Clinicaltrial.gov Registry, NCT03668262, Registered on 11 September 2018. © 2020 The Author(s).