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Background: Discrepancies in electrolyte concentration, namely sodium and potassium using the direct and indirect ion selective electrode (ISE) methods have been described. The present study was taken up to compare serum chloride values obtained using the direct and indirect ISE methods. Methods: Serum chloride measurement was done in residual samples received in the clinical laboratory using direct ISE method and indirect ISE method. Imprecision, accuracy and comparability of the methods were studied as per the Clinical and Laboratory Standards Institute EPA guidelines. The American Clinical Laboratory Improvement Amendments (CLIA) guideline of acceptability of target ±5% was considered as the quality goal. Results: Both the methods had acceptable imprecision and inaccuracy of <5%. Good agreement between the two methods was seen as assessed using Bland–Altman plot and Passing–Bablok regression analysis with a correlation coefficient of 0.9838. A significant difference in serum chloride levels measured in patient samples covering the entire analytical range was observed (P < 0.001), with the results of serum chloride by indirect ISE being lower compared to the direct ISE method. The difference between the two methods was below the target ±5% and thus met the CLIA guidelines. Conclusions: The findings of the present study show that serum chloride estimation using direct ISE and indirect ISE methods are comparable.