Simple Summary Near-infrared spectroscopy (NIRS) has emerged as an accurate and promising alternative to traditional wet chemistry methods in feed and food science. Its applicability extends to estimating concentrations of compounds such as n-alkanes, based on their chemical properties, in various materials, including feed and feces. Analysis of excreta n-alkane patterns can provide insight into the dietary behavior of laying hens, particularly in scenarios where they have access to free-range areas and potentially consume plants from outside sources. Our study attempts to explore extreme cases, such as hens consuming only commercial feed, which result in lower concentrations of n-alkanes in excreta, thus challenging NIRS as a replacement for wet chemistry. Evaluating the accuracy of NIRS in predicting n-alkanes in excreta is critical because of its potential for substantial time and cost savings. Furthermore, it contributes to a deeper understanding of future nutritional strategies for laying hens, particularly in light of external nutritional contributions.Abstract 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.