Background and Objectives: Organophosphate compounds are frequently used for homicidal and suicidal purposes, organophosphates account for as many as 80% of pesticide-related hospital admissions. This study was done to estimate serum amylase and plasma cholinesterase (ChE) in acute organophosphorus (OP) poisoning, to correlate serum amylase with plasma ChE levels and to study serum amylase levels as a probable prognostic marker in acute OP poisoning. Materials and Methods: The study was conducted at the hospitals attached to Bangalore Medical College and Research Institution, Bangalore, from November 2017 to May 2019, 110 cases of OP poisoning were selected based on inclusion and exclusion criteria. Patients were grouped accordingly based on ChE levels into mild, moderate, and severe. Serum amylase levels were estimated on admission, at 48 h, and at outcome either recovery or clinical deterioration. Serum amylase was used as a marker to assess severity in acute OP-poisoning cases, to predict ventilator requirement and mortality, and also for prognostication. Results: In our study, a total of 110 acute OP-poisoning cases were included. The mean serum amylase levels at admission, 48 h, and the outcome were 54.81, 54.44, and 53.35 among the nonintubated group, respectively, and 152.23, 152.67, and 141.13 among the intubated group, respectively, with a significant P value (0.000*). This shows that patients who were intubated had elevated mean serum amylase levels (>90 U/l) in comparison to patients who were not intubated. Sixty-three patients had normal amylase levels on day 1 (≤90 U/l) (normal value of serum amylase as per the laboratory was 28–90 U/l) and 47 patients had raised amylase levels on day 1 (>90 U/l). Among 47 patients with raised amylase level, 18 patients died and there were no deaths in the normal amylase level group with P = 0.00* which is statistically significant. Raised serum amylase correlated well in predicting ventilator requirement and mortality in patients with OP poisoning. Conclusion: Serum amylase can be used as a reliable biochemical marker as it is easily available, relatively cheap, and it also predicts the requirement for intubation and mortality in acute OP-poisoning cases. Increased amylase levels on admission imply poorer outcome and increased risk of mortality, and thereby, it can be used as an alternative marker to predict clinical outcome and for prognostication.