The objective of this study was to elucidate the mixing state of proteins and amino acid excipients concentrated in the amorphous non-ice region of frozen solutions. Thermal analysis of frozen aqueous solutions was performed in heating scans before and after a heat treatment. Frozen aqueous solutions containing a protein ( e.g. , recombinant human albumin, gelatin) or a polysaccharide (dextran) and an amino acid excipient ( e.g. , L-arginine, L-arginine hydrochloride, L-arginine monophosphate, sodium L-glutamate) at varied mass ratios showed single or double T _g′ (glass transition temperature of maximally freeze-concentrated solutes). Some mixture frozen solutions rich in the polymers maintained the single T _g′ of the freeze-concentrated amorphous solute–mixture phase. In contrast, amino acid-rich mixture frozen solutions revealed two T _g′s that suggested transition of concentrated non-crystalline solute–mixture phase and excipient-dominant phase. Post-freeze heat treatment induced splitting of the T _g′ in some intermediate mass ratio mixture solutions. The mixing state of proteins and amino acids varied depending on their structure, salt types, mass ratio, composition of co-solutes ( e.g. , NaCl) and thermal history. Information on the varied mixing states should be valuable for the rational use of amino acid excipients in lyophilized protein pharmaceuticals.