Background <p>Bronchial challenge testing with methacholine was applied to evaluate and quantify bronchial hyperresponsiveness (BHR) in asthma patients. We aimed to compare the clinical effectiveness between methacholine chloride and methacholine for methacholine challenge testing (MCT) in clinical practice, and investigate the adverse events associated with methacholine chloride.</p> Methods <p>Patients who received methacholine and inhaled methacholine chloride for MCT were included in this retrospective study. All participants completed pulmonary function tests and MCT between January 2022 and February 2024. The provocative dose of methacholine that results in a 20% fall in FEV₁ (PD20-FEV₁) was used as quantitative measure of bronchial hyperresponsiveness. The primary outcome of the study was the proportion of positive MCT results and the degree of BHR, while the secondary outcome was the incidence rate of respiratory adverse events during methacholine chloride administration.</p> Results <p>A total of 17,352 participants were included. In patients with bronchial symptoms, those administrated methacholine chloride for MCT demonstrated a significantly higher percentage of positive results compared with those administrated conventional methacholine (36.7% <i>vs.</i> 30.8%, <i>P</i> &lt; 0.001). Moreover, PD20-FEV₁ values were significantly lower in methacholine chloride group than methacholine group (<i>P</i> &lt; 0.001). Chest tightness was the most prevalent adverse symptom affecting 30.8% of patients who received methacholine chloride, followed by cough at 27.2%. No serious adverse events were reported in these patients.</p> Conclusions <p>The data indicated that methacholine chloride yielded a higher positive test rate than methacholine in MCT. Given that only mild adverse symptoms of chest tightness and cough were observed, with no serious adverse events reported, it represents a safe and effective alternative for clinical BHR assessment.</p>