Lead (Pb) is used in many industrial applications and is a toxic heavy metal that causes health hazards. Lichens are symbiotic associations that are environment friendly in removing toxic elements from the polluted environment. They can accumulate heavy metals and are therefore considered in biomonitoring of metal pollution. In this study, biochemical markers due to lead toxicity on Cladonia convoluta were revealed using two-dimensional correlation (2DCOS) spectra analysis. The 2DCOS enhances the resolution of the spectrum and gives more information on the interaction of functional groups within and between molecules which cannot be revealed from conventional spectra. Thus, 2DCOS is an efficient tool in monitoring even minute spectral changes in complex spectra. The results of conventional IR analysis showed changes in the band area. It follows in the order polysaccharides > proteins > lipids due to lead toxicity. The results of 2D correlation spectra show predominant changes at the pyranose ring. The hetero asynchronous map show correlation changes of pyranose ring with lipid and proteins. It reveals a change in carbohydrate synthesis due to toxicity. The highest positive and negative PCA loading values show deformation pyranose ring to glucan band. The results of HCA show samples are well separated into two major clusters with high separation values. ROC analysis gives good reliability of the test. Hence, 2DCOS coupled with chemometric analysis helps in understanding the biomolecular changes in C. convoluta effectively. Keywords: 2DCOS; Lichens; Polysaccharides; ROC; Pb.