A customized filtering technique is introduced and compared with fast Fourier transformation (FFT) for analyzing heart rate variability (HRV) in neonates from short-term recordings. FFT is classically the most commonly used spectral technique to investigate cardiovascular fluctuations. FFT requires stability of the physiological signal within a 300 s time window that is usually analyzed in adults. Preterm infants, however, show characteristics of rapidly fluctuating heart rate and blood pressure due to an immature autonomic regulation, resulting in non-stationarity of these signals. Therefore neonatal studies use (half-overlapping or moving) windows of 64 s length within a recording time of 2–5 min. The proposed filtering technique performs a filtering operation in the frequency range of interest before calculating the spectrum, which allows it to perform an analysis of shorter periods of only 42 s. The frequency bands of interest are 0.04–0.15 Hz (low frequency, LF) and 0.4–1.5 Hz (high frequency, HF). Although conventional FFT analysis as well as the proposed alternative technique result in errors in the estimation of LF power, due to spectral leakage from the very low frequencies, FFT analysis is more sensitive to this effect. The response times show comparable behavior for both the techniques. Applying both the methods to heart rate data obtained from a neonate before and after atropine administration (inducing a wide range of HRV), shows a very significant correlation between the two methods in estimating LF and HF power. We conclude that a customized filtering technique might be beneficial for analyzing HRV in neonates because it reduces the necessary time window for signal stability.