Mass spectrometry (MS) based proteomic studies on dendritic cells (DCs) facilitate the understanding of the molecular processes occurred in the cells. In order to support the investigation of DC protein expression during anti-viral response to HIV-1 attack, three key steps, sample preparation, MS data acquisition and data analysis, in shotgun proteomic workflow were investigated. The final established high resolution MS proteomic workflow employed a gel-free and MS-friendly sample preparation procedure and an optimized data-dependent acquisition (DDA) method implementing the concept of exclusion/inclusion list for enhanced protein identification and a variable window strategy in SWATH MS data acquisition for improved proteomic qualitative and quantitative analysis. Comparison of the proteomic profiles of DCs after different treatments revealed 139 proteins involved in the viral-restrictive state established by bacterial lipopolysaccharide (LPS) and 16 and 23 novel candidate proteins that are putatively associated with the Vpx enhanced HIV-1 infection of immature and mature DCs respectively.