The determination of conjugated linoleic acids (CLA) in cow milk fat was studied by using UV (210-250 nm) and Fourier transform (FT)-Raman (900-3400 cm (-1)) spectroscopy in order to determine the best spectrophotometric technique for routine analysis of milk fat. A collection of 57 milk fat samples was randomly divided into two sets, a calibration set and a validation set, representing two-thirds and one-third of the samples, respectively. All calculations were performed on the calibration set and then applied to the validation set. The CLA content ranged from 0.56 to 4.70%. A comparison of various spectral pretreatments and different multivariate calibration techniques, such as partial least-squares (PLS) and multiple linear regression (MLR), was done. This paper shows that UV spectroscopy is as reliable as FT-Raman spectroscopy to monitor CLA in cow milk fat. The best calibration for FT-Raman was given by a PLS model of seven factors with a standard error of prediction (SEP) of 0.246. For UV spectroscopy, PLS models were also better than MLR models. The most robust PLS model was constructed with only one factor and with SEP=0.288.