This study explored the potential of Raman spectroscopy for the analysis of poly(3-hydroxybutyrate) (PHB) in bacteria. PHB can be formed in large amounts by certain bacteria as a storage material and is of high importance for industrial biodegradable plastic production. Raman spectra were collected from Cupriavidus necator DSM 428 (H16), from its non-PHB-producing mutant strain C. necator DSM 541, and from pure PHB, in order to determine at which Raman shifts a contribution of PHB in bacterial spectra can be expected. The Raman band intensity at ca. 1734 cm-1 appeared to be suitable for the monitoring of PHB production and consumption. These intensities were linearly related to the PHB concentration (mg L-1 culture) determined by parallel HPLC analysis. Therefore, Raman spectroscopy is considered as a fast and noninvasive technique for the determination and monitoring of the PHB content in bacteria.