Speech emotions have potential to provide valuable source of information which can lead us toward human perception and decision making process. This paper analyzes the variation and effect on prosodic features (Formant and Pitch) of female and male speakers in two different emotions (angry and neutral) and softwares (PRAAT and MATLAB) in Urdu language using two ways ANOVA testing. The objective of this paper is to determine the significant effect of emotions and softwares on prosodic features (Pitch and Formant) using recorded speech emotion of both male and female voices of same age group in Urdu language. Experimental results of two-way ANOVA testing considerably show that emotions have effect on pitch and formant both in male and female voice unlike software.