Linoleic acid is a polyunsaturated omega-6 fatty acid found as phytoconstituent in many plant species. It is reported to have activity such as 5-alpha-reductase-inhibitor, antianaphylactic, antiarthritic, ntiatherosclerotic, anticancer, anticoronary, antieczemic, antifibrinolytic and many others. In this research work we have developed a method by HPTLC to identify and standardize linoleic acid in methanolic extract of Solanum nigrum berries. Calibration curve of Linoleic acid was plotted and was found to have r2 value of 0.99451. The percentage content of Linoleic acid in Solanum nigrum berries methanolic extract was found to be 9.32% w/w.