In this work, we investigate the feasibility of multi-wavelength ultra-violet spectroscopy for the quantification of β-lactoglobulin and α-lactalbumin in ultrafiltration permeates from protein fractionation processes. Spectra from solutions of pure proteins were compared and distinctive characteristics for the two proteins were identified. Subsequently, two different calibration approaches were tested to overcome the “cage of covariance” that is inherent in protein fractionation and up-concentration processes. Selection of wavelength regions allowed for the prediction of the β-lactoglobulin and α-lactalbumin concentration with high precision and accuracy, reaching a root mean square error of cross-validation of 0.26 w/w% (concentration range 0–10 w/w%∗) protein for α-lactalbumin and 0.11 w/w% (0–10 w/w%) protein for β-lactoglobulin. This proves the potential of the methods developed for implementation as rapid monitoring of protein composition in permeates from ultrafiltration processes. The developed Partial Least Squares (PLS) regression models were used to predict protein composition in a continuous mode during two lab-scale filtration experiments. The results obtained show that UV spectroscopy can be used, along with tailored chemometrics techniques, for monitoring protein composition in protein fractionation processes both at-line and potentially in-line.