Introduction Brain oedema is a major complication after craniotomy. Hyperosmolar agents have been used as the medical treatment for this condition. Measurement and estimation of serum osmolality during hyperosmolar agent infusion is of clinical importance to evaluate clinical efficacy, adjust dosage and avoid side effects. However, several studies have shown that calculated serum osmolality may lead to a systematic bias compared with direct measurement. In the present study, mannitol or hypertonic saline (HS) will be used in patients after elective craniotomy. We aim to determine the accuracy of serum osmolality estimation during the application of hyperosmolar agent. Methods and analysis The study is a prospective, randomised, double-blinded, controlled, parallel-group design. Adult patients requiring the use of hyperosmolar agents for the prevention or treatment of postoperative brain oedema are enrolled and assigned randomly to one of the two treatment study groups, labelled as M group' and HS group'. Patients in the M and HS groups receive intravenous infusion of 125mL of either 20% mannitol or 3.1% sodium chloride solution, respectively. Data will be collected immediately before the infusion of study agents, 15, 30, 60, 120, 240 and 360min after the start of infusion of experimental agents, which includes serum osmolality, concentration of serum sodium, potassium, urea and glucose. Serum osmolality will be measured by means of freezing point depression. Estimated serum osmolality will also be calculated by using four formulas published previously. Osmole gap is calculated as the difference between the measured and the estimated values. The primary endpoint is the correlation of measured and estimated serum osmolality during hyperosmolar agent infusion. Ethics and dissemination The study was approved by the International Review Board (IRB) of Beijing Tiantan Hospital, Capital Medical University. Study findings will be disseminated through peer-reviewed publications and conference presentations. Trial registration number ClinicalTrials.gov identifier: NCT02037815.