The derivative of potential energy with respect to the coupling parameter (dU/dλ) emerged as a highly sensitive probe of timestep-induced artifacts (Tables ). While timesteps ≤ 2 fs maintained consistent dU/dλ values (± 0.5 kcal/mol) across λ states, 4 fs timesteps produced deviations up to 3 kcal/mol in systems with large softcore transformations. Statistical analysis through t-tests (Tables ) revealed that 85% of comparisons between 4 fs and 0.5 fs timesteps showed significant statistical differences (p < 0.05), compared to only 15% at 2 fs. These findings challenge the notion that thermostatting in NPT ensembles mitigates timestep effects, as significant discrepancies persisted even with Langevin dynamics applied.