Acid-base disturbances have been usually evaluated with the traditional Henderson-Hasselbach method and Stewart's physiochemical approach by quantifying anions of tissue acids (TA). It is hypothesized that an increase in tissue acids during metabolic acidosis would cause a compensatory decrease in the plasma chloride (Cl) relative to sodium (Cl-Na ratio) in order to preserve electroneutral balance. Therefore, we aimed to investigate the use of Cl-Na ratio as a bedside tool to evaluate the identifying raised TA in neonates as an alternative to complex calculations of Stewart's physiochemical approach. This retrospective study was conducted between January 2008 and December 2009. Infants were included in the study when blood gas analysis reveals a metabolic acidosis; pH <7.25 and sHCO(3) concentration was <22 mEq/L. The Cl-Na ratio, sodium-chloride difference (Diff(NaCl)), anion gap (AG), albumin-corrected AG (AG(corr)), strong ion difference (SID), unmeasured anions (UMA), and TA were calculated at each episode of metabolic acidosis. A total of 105 metabolic acidosis episodes occurred in 59 infants during follow-up. Hypochloremic metabolic acidosis occurred in 17 (16%) of samples, and all had increased TA. The dominant component of TA was UMA rather than lactate. There was a negative correlation between the Cl-Na ratio and SID, AG(corr), UMA, and TA. Also, there was a positive correlation between Diff(NaCl) and SID, AG(corr), UMA, and TA. Base deficit and actual bicarbonate performed poorly in identifying the TA. In conclusion, our study suggested that Diff(NaCl) and Cl-Na ratio are simple and fast, and may be an alternative method to complex Stewart's physiochemical approach in identifying raised UMA and TA in critically ill neonates.