Core Ideas Critical S dilution curve was established for maize: S concentration = 2.13 × biomass−0.23. The S nutrition index successfully diagnosed S status at early crop stages (∼V6). Sulfur concentration and chlorophyll meter reading, both in the upper leaf, were good predictors of the S nutrition index. Sulfur (S) deficiency can severely limit maize (Zea mays L.) yield. This deficiency could be predicted by quantifying the S concentration (SC) in the shoot biomass (BM) and by calculating the S nutrition index (SNI = observed SC/critical SC). However, as shoot SC determination is laborious, alternative S diagnostic methods should be developed in maize. Thus, the objectives of our study were to: (i) determine and validate a critical S dilution curve, (ii) quantify the critical SNI, and (iii) explore the use of SC and chlorophyll meter reading (CMR), both determined in the uppermost developed leaf at vegetative growth stages, to predict SNI and for diagnosing S status in maize. Six field studies evaluating fertilizer S rates were executed, collecting maize shoot and uppermost developed leaf samples and determining Sc, CMR, yield, and SNI parameters. The main outcomes from our study were: (i) a maize critical S dilution curve was fitted (SC = 2.13BM−0.23) and validated with an independent dataset; (ii) a critical SNI threshold of 0.79 adequately diagnosed S status at ∼V6 stage; (iii) at V6, SNI was linearly related to SC (R2 = 0.65) and CMR (R2 = 0.85). As a result, a threshold value of 2.0 g S kg−1 for SC, 47.5 units for CMR, and 0.94 units for relative CMR can be used to successfully diagnose S deficiencies at early vegetative (∼V6) maize stages. Future studies testing S diagnostic tools for maize should be performed in environments with severe S deficiency and nutrient co‐limitations.