Protein solubility, ruminal degradation and intestinal digestibility are strongly related to their inherent molecular makeup. This study was designed to quantitatively evaluate protein digestion in the rumen and intestine of dairy cattle, and estimate the content of truly metabolizable protein (MP) in newly developed cool-season forage corn cultivars. The second objective was to quantify protein inherent molecular structural characteristics using advance molecular spectroscopic technique (FT/IR-ATR) and correlate it to protein metabolic characteristics. Six new cool-season corn cultivars, including 3 Pioneer (PNR) and 3 Hyland (HL), coded as PNR-7443R, PNR-P7213R, PNR-7535R, HL-SR06, HL-SR22, HL-BAXXOS-RR, were evaluated in the present study. The metabolic characteristics, MP supply to dairy cattle, and energy synchronization properties were modeled by two protein evaluation models, namely, the Dutch DVE/OEB system and the NRC-2001 model. Both models estimated significant (P<0.05) differences in contents of microbial protein (MCP) synthesis and truly absorbable rumen undegraded protein (ARUP) among the cultivars. The NRC-2001 model estimated significant (P<0.05) differences in MP content and degraded protein balance (DPB) among the cultivars. The contents MCP, ARUP and MP were higher (P<0.05) for cultivar HL-SR06, resulting in the lowest (P<0.05) DPB. However, none of the cultivars reached the optimal target hourly effective degradability ratio [25gNg/kg organic matter (OM)], demonstrating N deficiency in the rumen. There were non-significant differences among the cultivars in molecular-spectral intensities of protein. The amide I/II ratio had a significant correlation with ARUP (r=-0.469; P<0.001) and absorbable endogenous protein (AECPNRC) (P<0.001; r=0.612). Similarly, amide-II area had a weak but significant correlation (r=0.299; P<0.001) with RUP and ARUP, and with AECPNRC (P<0.001; r=0.411). Except total digestible nutrients and AECPNRC, the amide-I area did not show significant correlations with DVE/OEB and NRC predicted protein fractions. This study shows that molecular spectroscopy can be potentially used as a rapid tool to quantify protein molecular makeup and screen the protein nutritive value of forage corn.