In present study the RP-HPLC characteristics were used for the evaluation of insulin interactions. These characters such as retention time (RT), area under the peak (AP), peak height (PH), and peak width (PW) were all examined. Free or uncontrollable cholesterol (UC) and fenugreek extract (FG) have been chosen to be the fat and food tested metabolites. It seems, as a general conclusion, that food fragment is considered to be preventive agent more than to be a treatment, meanwhile, it is a pH value dependent interaction, i.e. more acid or base in blood is less responded by food metabolites. The most important achievement in present study was the capability ol the technique to specifically determine the insulin interaction with UC. The new sharp < /p> analytical measurement is centered in a new peak of RT of more than 4 min for the protein only connected with the presence of UC. The new peak for the hormone is another one than its preceding original peak of about 2 min. The other mentioned HPLC characters sensitivity to these particular interactions was determined and separately discussed. A modified technique based on this fast and simple assay• may add a nutritional and medical significance to the field of cholesterolemia, hence insulin interaction becomes one of the most important molecular biology diagnostic test that constitute cholesterolernia cluster factors. These reactions were strongly recorded in both chromatographic and chromophorici elements, especially AP, PW and PH. Moreover, the HPLC data has been supported by some spectrophotometric techniques.