Display omitted •Chemical profiling and network pharmacology-based analysis of JWQFY were performed.•A total of 135 constituents were identified, 70 of them were further quantified.•Multi-target mechanisms of JWQFY against Alzheimer's disease were predicted.•The modification rationality of the formula JWQFY was elucidated. Jia-Wei-Qi-Fu-Yin (JWQFY) is a newly developed anti-Alzheimer's disease (AD) prescription modified from a classical traditional Chinese medicine formula, Qi-Fu-Yin (QFY). However, a systematic understanding of its chemical constituents and molecular mechanisms is still elusive. To address this problem, comprehensive chemical profiling followed by network pharmacology-based analysis of JWQFY was performed. Firstly, a total of 136 compounds were characterized by high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (HPLC-QTOF MS), 17 of them were specifically identified in JWQFY comparing with QFY. Seventy compounds were further quantified via a validated HPLC coupled with triple quadrupole tandem mass spectrometry (QQQ MS) method. Then the protein targets of the seventy compounds were gathered from public databases for network construction. As a result, fifty-seven compounds were filtered, which interacted with 655 targets. Thirty-four of them were mapped into the KEGG pathway of AD, indicating JWQFY might exert anti-AD effects by anti-inflammation, neuronal apoptosis intervening, Aβ production inhibition and phosphorylating tau protein moderating. Furthermore, in the compound-target-AD network, a list of hub compounds and hub targets was identified based on their topological features, including the degree, node betweenness and closeness. Four of the hub compounds were specifically originated from JWQFY, supporting the modification rationality of this formula. This study provided a scientific basis for understanding the bioactive compounds and the multi-target mechanism of JWQFY.