Medical biophotonic tools provide new sources of diagnostic information regarding the state of human health which are used in managing patient care. In current study, Raman spectroscopy together with chemometric technique has successfully been demonstrated for screening of asthma disease. Raman spectra of sera samples from asthmatic patients as well as healthy (control) volunteers have been recorded at 532 nm excitation. In healthy sera, three highly reproducible Raman peaks assigned to β-carotene have been detected. Their sensitive detection is facilitated due to the resonance Raman effect. In contrast, in asthmatic patients sera, the peaks assigned to β-carotene are either diminished or suppressed accompanied by other new Raman peaks. These new peaks most probably arise due an elevated level of proteins, which could be used to identify/differentiate between asthma and non-asthma samples. Furthermore, partial least squares discrimination analysis (PLS-DA) model was developed and applied on the Raman spectra of diseased as well as healthy samples which successfully classify them. The correlation coefficient (r2) of the model was determined as 0.965. Similarly, the root mean square errors in cross-validation (RMSECV) and in prediction (RMSECP) are 0.09 and 0.25 respectively. PLS-DA has the potential to be incorporated in a microcontroller’s code, attached with a hand-held Raman spectrometer, for screening purposes in asthma, which is a disease of great concern for the clinicians especially in children.