This study introduces an approach to enhance the Raman calibration model cross‐scale prediction capabilities in cell cultures. Our investigation centers on the improvement of Raman calibration models along with the scaling‐up of mammalian cell culture processes. Initially, we observed that integrating data from a 50 L run into the original dataset at lab‐scale was an effective strategy for improving prediction accuracy on 1000 L run. Further investigation indicated that this improvement could be attributed to the similarity between the spectra of 50 L with 1000 L. Subsequent investigation unveiled that the spectral variations primarily stemmed from differences between the instruments. This was verified through a direct comparison of two Raman analyzers. Then two pairwise spectral transformation methods, direct standardization (DS) and piecewise DS, both exhibited superior performance in predicting. This research underscores the importance of Raman spectral transfer method to alleviate instrument‐to‐instrument variations in cross‐scale monitoring of cell culture processes.