Influenza viruses, characterized by antigenic drift and shift, can cause seasonal epidemics or pandemics. According to World Health Organization (WHO) data, approximately 650,000 global annual deaths are attributed to influenza-associated respiratory diseases, highlighting the urgent need for vaccine protection in high-risk populations. Residual host cell protein (HCP) may elicit allergic reactions or compromise antigen stability, with their permissible limits stringently regulated by international health authorities. This study employed liquid chromatography-tandem mass spectrometry (LC-MS) to systematically evaluate a downstream purification process involving ultrafiltration concentration coupled with Capto? Core 700 multimodal chromatography and anion-exchange chromatography (AEC). Key results demonstrated that ultrafiltration concentration of the viral harvest significantly reduced HCP content, achieving a removal rate of 90.32%. Subsequent purification steps further eliminated HCP, with gel filtration chromatography and AEC providing removal rates of 92.47% and 61.52%, respectively. Concurrently, the principal antigen hemagglutinin (HA) increased from 2.21% to 8.61% in the viral bulk. HCP residuals were reduced to 535.67 ng per dose, while residual recombinant nuclease fell below the detection limit of 0.312 ng/mL. Proteomic profiling indicated substantial changes in dominant HCP species following ultrafiltration, with Annexin A2 (ANXA2), Actin cytoplasmic 2 (ACTG), and IF rod domain-containing protein predominating in the concentrate. The subsequent purification maintained consistent HCP profiles, primarily composed of ANXA2, ACTG, and Peroxiredoxin-1 (PRDX1). Overall, ultrafiltration critically reduces HCPs and process-related impurities, while multimodal chromatography effectively enriches key antigens (HA/NA). The established LC/MS platform might provide a robust analytical framework for influenza vaccine quality control and process optimization.