In the case of alfalfa, the methodology deals with the assessment of the influence of location, cutting order and weather in five consecutive years (2018–2022) on the content of nitrogenous substances, fiber and ash. The main goal of the research was to obtain data for the calibration of instruments in near infrared spectroscopy (NIRS) for fresh and wilted alfalfa. 1080 alfalfa samples were included in the statistical evaluation, half of them for chemical analysis and half for NIRS analysis. The results show that factors such as the development of the weather, the location and the sequence of mowing can affect the content of dry matter and the contents of individual nutrients. Compared to NIRS, chemical analysis determined higher contents of dry matter (by 9 g/kg dry matter), detergent fiber acid (by 10.2 g/kg dry matter) and ash (2.2 g/kg dry matter) and lower N-substances (by 11.2 g/kg dry matter).