Negative energy balance (NEB) is a common and difficult to solve problem in dairy herds. NEB mostly affects in dairy cows after calving and in the first phase of lactation, when dairy cows are unable to take in sufficient dry matter to meet the energy requirements for high lactation. The negative impacts of NEB on the health, production and reproductive of dairy cows have challenged farmers and the research community to find ways to estimate it early. Dairy cows in NEB can be identified by measuring blood concentrations of non-esterified fatty acids as standard, but this method requires repeated blood sampling and subsequent financial, organisational and time-consuming laboratory analysis, so this method is not used in practice. The aim of the methodology was described to use of indirect indicators of NEB, which are relatively easily and cost-effectively available and can detect NEB in early lactating dairy cows with high reliability. The methodology exploits change in the content of some milk components, especially fatty acids (FAs). Obtaining information about FAs concentrations through extended milk analysis in an accredited laboratory for milk analysis in the under of milk recording a completely new concept, which has not been applied in the Czech Republic so far, together with the use of ROC curves (sensitivity and specificity) and the subsequent possibility of determining threshold values of some FAs for the conditions of a specific herd.