Raman optical activity (ROA) is an incisive probe of structural dynamics and its combination with two-dimensional correlation analysis is an attractive method for the investigation of conformational transitions in proteins. In this paper we show that pretreatment of data is essential for the generation of reliable 2D ROA and 2D Raman correlations. Specific experimental parameters and available information of ROA and Raman studies have facilitated the development of four pretreatment processes, baseline subtraction, smoothing, normalization and further baseline correction. The bisignate nature of ROA spectra has a significant effect on the interpretation of synchronous 2D correlation plots when bands at wavenumbers of both positive and negative intensity are compared, while interpretation of asynchronous plots remains unchanged. We introduce a notation format to make interpretation of 2D correlations from bisignate spectra clearer. With appropriate application of 2D correlation analysis to ROA spectra, 2D ROA and 2D Raman/ROA heterocorrelation are potentially valuable tools for the analysis of conformational transitions in polypeptides and proteins. (c) 2006 Elsevier B.V. All rights reserved.