Andrographis paniculata is known for therapeutic potentials including antiviral, antimicrobial, anticancer, antineoplastic, and anti-inflammatory properties. Flavonoids and diterpenoids of A. paniculata have been extensively studied for their anticancer and anti-inflammatory effects. Although extracts of Andrographis paniculata have shown anti-breast cancer activity, the molecular targets of its medicinal molecules remain unclear. This study aimed to identify the anti-breast cancer molecules of Andrographis paniculata and investigate their underlying mechanisms. In silico screening and docking studies were conducted to identify active anticancer compounds, followed by in vitro mechanistic validation. Bioactive compounds targeting PI3K were identified, with the diterpenoid neoandrographolide exhibiting superior binding affinity compared to the reference inhibitor alpelisib. Additionally, andrographolide and neoandrographolide demonstrated high gastrointestinal absorption in ADME studies. In vitro assays using PI3K-expressing MDA MB-231 cells showed that neoandrographolide exhibited excellent anticancer effects, inducing programmed cell death and arresting proliferation in the G1 phase by downregulating PI3K expression. This study identifies neoandrographolide as a strong PI3K-targeting compound with potential anticancer activity against triple-negative breast cancer (TNBC). © 2023 Elsevier B.V., All rights reserved.