Low-cost potentiometric and spectrophotometric procedures for cephalothin (CPI) determination in pure and biological fluids were investigated. The potentiometric technique is created through titration of CPI with an aqueous medium of 0.1 M NaOH at an ionic strength of μ = 0.3 M sodium chloride and room temperature by a combined glass pH electrode. Using the standard addition method, we found that the detection and quantitative limits were 0.042 mg/mL, with the standard deviation SD = 0.011, correlation coefficient R = 0.9880 (n = 5), and linear concentration ranges from 0.042 to 0.82 mg/mL. This technique was utilized to assess CPI in pure solutions, urine, and serum with suitable results. No interference was exposed in the presence of public components of the samples under study. Recovery of CPI for pure and biological fluids is in the range of 98.2-101%. Also, the spectrophotometric method has been performed through the formation of the Prussian Blue (PB) complex. The reaction between the acidic hydrolysis product of CPI (T = 60 °C) and the mixture of Fe3+ with hexacyanoferrate (III) ions (HCF(III)) was detected for the spectrophotometric determination of the drug. The maximum absorbance of the formed complex was measured at λ = 283 nm with 2.0 × 103 L mol-1 cm-1 molar absorptivity. Reaction states have been advanced to acquire the PB complex of great sensitivity and longer stability. In optimal states, the absorbent of the PB compound was attained to grow linearly with the increase in the concentration of CPI, which agrees with the correlation coefficient values. The detection and quantitative limits were 0.000036 and 0.0012 mg/mL, respectively, with the standard deviation, SD = 0.0005, correlation coefficient, R = 0.9955 (n = 5), and the linearity range of the calibration plot 0.0005-0.02 mg/mL CPI. The planned technique was positively utilized for the detection of CPI in both urine and serum models. The results fit well with the data found from the potentiometric method.