The invention belongs to the field of analytical chemistry, and relates to a method for determining the content of benzalkonium chloride in a salmon calcitonin nasal spray by an HPLC method. A chromatographic column adopted in the method adopts octadecyl silane chemically bonded silica as filler, and an optimized mobile phase A and a mobile phase B are adopted for isocratic elution; and a drug isa polypeptide nasal spray, and a bacteriostatic agent is benzalkonium chloride. According to the method, the content of the bacteriostatic agent in the salmon calcitonin nasal spray of polypeptide drugs can be conveniently and accurately determined; and the method is easy and convenient to operate, rapid, accurate, high in sensitivity, good in repeatability, mild in condition and small in trailingfactor, through systematic methodology verification, it is proved that the method is applied to the determination of the content of the benzalkonium chloride in the salmon calcitonin nasal spray, theinspection results are accurate and reliable, and great significance to the quality control of the salmon calcitonin nasal spray is achieved.