Obesity has consistently presented a significant challenge, with excess body fat contributing to the development of numerous severe conditions such as diabetes, cardiovascular disease, cancer, and various musculoskeletal disorders. In this study, different methods are proposed to study substrate utilization (carbohydrates, proteins, and fats) in the human body and validate the biomarkers enabling to investigation of weight management and monitor metabolic health. The first technique to study was Indirect calorimetry, which assessed Resting Energy Expenditure (REE) and measured parameters like oxygen consumption (VO2) and carbon dioxide production (VCO2). A validation study was conducted to study the effectiveness of the medical device Breezing Med determining REE, VO2, and VCO2. The results were compared with correlation slopes and regression coefficients close to 1. Indirect Calorimetry can be used to determine carbohydrate and fat utilization but it requires additional correction for protein utilization. Protein utilization can be studied by analyzing urinary nitrogen. Therefore, a secondary technique was studied for identifying urea and ammonia concentration in human urine samples. Along this line two methods for detecting urea were explored, a colorimetric technique and it was validated against the Ion-Selective method. The results were then compared by correlation analysis of urine samples measured with both methods simultaneously curves. The equations for fat, carb, and protein oxidation, involving VO2, VCO2 consumption, and urinary nitrogen were implemented and validated, using the above-described methods in a human subject study with 16 subjects. The measurements included diverse diets (normal vs. high fat/protein) in normal energy balance and pre-/post interventions of exercise, fasting, and a high-fat meal. It can be concluded that the indirect calorimetry portable method in conjunction with urine urea methods are important to help the understanding of substrate utilization in human subjects, and therefore, excellent tools to contribute to the treatments and interventions of obesity and overweighted populations.