Since the yeast Phaffia rhodozyma was first described some 35 years ago, there has been significant interest in the development of commercial processes to exploit its ability to produce carotenoids (∼80% astaxanthin). However, the optimal conditions for carotenoid production are not well understood. A key limitation has been the lack of an appropriate sensor for on-line carotenoid quantification. In this study, an in situ Raman spectroscopy probe was used to monitor intracellular carotenoid production for three consecutive P. rhodozyma fed-batch experiments. Raman spectroscopy is particularly well suited to the study of carotenoids due to a resonance effect, which greatly enhances the intensity of the three fundamental carotenoid bands, ν1 (1513 cm−1, CC stretch), ν2 (1154 cm−1, C—C stretch), and ν3 (1003 cm−1, CH3 rock). For all three cultures, the peak height of these bands was linearly correlated with intracellular carotenoid content (1 to 45 mg/L) to a precision of better than 5%, and the correlation from one experiment was directly applicable to others. ©2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 668–680, 2003.