N-alkanes offer a promising approach for assessing the nutritional contribution of external sources to the diets of laying hens in free-range production systems. However, traditional laboratory methods, involving extraction, purification and gas chromatographic analysis, are both economically burdensome and time-consuming. Near-infrared spectroscopy (NIRS) is emerging as a viable alternative, with varying degrees of accuracy depending on the chemical nature and concentration of the component of interest. In our research, we focus on the accuracy of NIRS in predicting the concentrations of n-alkanes (C25–C33) in excreta under simulated free-range conditions with two different diets: one containing a commercial feed with minimal n-alkane content and another containing 1% alfalfa on top of the commercial feed. Spectra processing and calibration were tailored for each n-alkane, with NIRS performance influenced by diet type. Notably, plant predictions using NIR-generated data were consistent with laboratory results, despite a slight tendency toward overestimation (3.40% using the NIRS-generated C25-C29-C33 combination versus 2.80% using laboratory analysis). This indicates the potential of NIRS as an efficient tool to assess n-alkanes in excreta of laying hens and, consequently, the nutritional contribution of the free-range environment, providing rapid and cost-effective results.