This study presents a portable spectroscopic system compatible with ambient light to assess hepatic steatosis (HS) and macrovesicular steatosis (MaS) in human liver specimens. Traditional assessment methods for MaS are limited, prompting the need for non-invasive alternatives. The study utilized a two-stage approach on thawed snap-frozen liver specimens. Biochemical validation compared fat content from Raman and reflectance intensities with triglyceride (TG) quantifications, while histopathological validation contrasted Raman-derived fat content with pathologist evaluations and an algorithm. Analysis of 16 specimens showed a positive correlation between spectroscopic data and TG quantifications. The Raman system differentiated various degrees of global HS and MaS in an additional 66 specimens. A dual-variable prediction algorithm classified significant discrepancies (≥10%) between algorithm-estimated global HS and pathologist-estimated MaS. This study demonstrates the viability of a portable spectroscopic system for non-invasive HS and MaS assessment to enhance real-time donor liver assessments during recovery to improve transplantation outcomes.