Determination of optimum sperm requirement in artificial breeding helps to improve the fertilization efficiency, sperm economy and minimize the sacrificing of males for milt. Quantification of sperm cells was done by spectrophotometer at 420 nm followed by haemocytometer counting. Best correlation (r2 = 0.97) was recorded. The minimum number of sperm required for optimal fertilization success in Heteropneustes fossilis was determined. An average fertilization success of 78–93% was recorded at 8 × 103 to 8 × 107 sperm per egg. The optimum contact duration of gametes was 5 minutes. The highest fertilization rate of 98.18% was recorded at 8 × 107 spermatozoa.egg−1.