A simple and intuitive formula for the estimation of urine osmolality (Uosm) using the measured concentrations of major active urine osmolytes over a wide range of urine dilutions was proposed in healthy cats. Sixty-three urine samples were retrieved using ultrasound-guided cystocentesis from sixteen healthy cats under 5 years of age receiving intravenous infusion over a period of 24 h. Samples were collected at baseline (T0), T2, T6, T12, and T24. Urine osmolality was measured using a freezing-point osmometer, and the concentrations of osmolytes (urea, sodium, glucose, and potassium) were evaluated. A simple linear regression model for a clinical use was selected, and the agreement between the calculated and actual urine osmolalities was assessed. Urinary concentrations of urea, sodium and glucose were the three variables included in the model with the lowest AICC. Urine osmolality can be predicted accurately and precisely using urine urea, sodium and glucose with the following equation: Uosm = 1.25 × urea (mmol/l) or 20.87 × urea (g/l) + 1.1 × sodium (mmol/l) + 67 × glucose (mmol/l) or 3.72 × glucose (mg/dl). The concordance correlation coefficient for repeated measures between the actual and the calculated urine osmolality was extremely close to 1, which supported a high agreement: 0.996 (CI 95%: [0.993; 0.998]). In a population of healthy cats, urine osmolality can be predicted accurately and precisely using urinary urea, sodium and glucose concentrations. Similar formulae could potentially be established to help the clinician in pathological situations