The aim of the present study was to develop the HPTLC fingerprint and to evaluate the in-vitro antioxidant, anti-inflammatory and anti-diabetic activities of ethanol extract of leaves of Actinodaphne madraspatana Bedd (A. madraspatana). HPTLC fingerprint analysis of ethanol extract was investigated by win CATS planar chromatography. The antioxidant activity was evaluated by the various antioxidant assays, such as total antioxidant, reducing power, DPPH radical scavenging, hydrogen peroxide scavenging and hydroxyl radical scavenging. The antioxidant activity was compared to standard drug ascorbic acid. In-vitro anti-inflammatory activity was evaluated by inhibition of albumin denaturation assay using aspirin as a standard drug. In-vitro anti-diabetic activity was evaluated by á-amylase inhibition assay using acarbose as a standard drug. The HPTLC fingerprint of ethanol extract has shown six peaks, which indicate the presence of six different phytocomponents. The in-vitro antioxidant, anti-inflammatory and anti-diabetic activities of ethanol extract of plant leaves increased with the increasing of concentration. The result revealed that extract in all the concentration showed the in-vitro antioxidant, anti-inflammatory and anti-diabetic activities compared to standard drugs. The ethanol extract of leaves of A. madraspatana has a potential effect as an antioxidant, anti-inflammatory and antidiabetic in all the tested in-vitro methods.