Advanced glycation end products (AGE) are produced by non-enzymatic reaction between glucose and biomolecules including proteins. AGE accumulation is known to cause alternations of structure and function in proteins and to be related with an increased risk of diabetic complications, cardiovascular diseases, and aging processes. Conventionally, AGE accumulation has been estimated by measuring auto fluorescence level using ultraviolet (UV) light excitation. In this study, we investigated an alternative approach to estimate auto fluorescence level and thus AGE accumulation in in vivo human skin using NIR (Near-Infrared) spectroscopy. To examine spectral features attributed to glycation in proteins, we first analyzed in vitro NIR spectra from native and glycated protein. Then, we further examined NIR spectra of in vivo skin from human subjects, and estimated their auto fluorescence level using several multivariate regression approaches. Our analysis in in vitro spectra from native and glycated albumin revealed that glycation may affect -CH and -NH stretching. Furthermore, we elucidated that those bands for -CH and -NH may be responsible for the variation in auto fluorescence level in human skin NIR spectra. Finally, auto fluorescence level was estimated from those NIR spectra using several multivariate regression methods: principal component regression (PCR), partial least square regression (PLS-R) and support vector regression (SVR). Among the three methods, SVR showed the best performance. We demonstrated in this study that NIR spectroscopy can be used as an alternative non-invasive method to estimate AGE accumulation in in vivo human skin tissue without UV radiation on skin tissue.