The purpose of this research was to develop rapid and cost-effective method for oestrus detection in dairy cows by means of near infrared spectroscopy and aquaphotomics, using raw milk from individual cows. We found that aquaphotomics approach showed consistent specific water spectral pattern of milk at the oestrus periods of the investigated Holstein cows. Characteristic changes were detected especially in foremilk collected at morning milking. They were reflected in calculated aquagrams of milk spectra where distinctive spectral pattern of oestrus showed increased light absorbance of strongly hydrogen-bonded water. Results showed that monitoring of raw milk near infrared spectra provides an opportunity for analysing hormone levels indirectly, through the changes of water spectral pattern caused by complex physiological changes related to fertile periods.